Methods of treating vascular inflammatory disorders

ABSTRACT

Provided are methods of treating or delaying the onset of a vascular inflammatory disease (e.g., acute lung injury) in a subject including administering to the subject a therapeutically effective amount of a nucleic acid containing all or a part of the sequence of mature miR-181b (SEQ ID NO: 1). Also provided are methods of decreasing nuclear factor-κβ (NF-κβ) signaling in an endothelial cell including administering to the subject a nucleic acid containing all or a part of the sequence of mature miR-181b (SEQ ID NO: 1).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/583,963, filed on Sep. 11, 2012, which is a U.S. National PhaseApplication under 35 U.S.C. §371 of International Application No.PCT/US2011/027772, filed on Mar. 9, 2011, which claims the benefit ofU.S. Provisional Patent Application Ser. No. 61/313,274, filed on Mar.12, 2010, the entire contents of each of which are hereby incorporatedby reference.

TECHNICAL FIELD

Described herein are methods for treating or delaying the onset of avascular inflammatory disease in a subject and decreasing nuclearfactor-κB (NF-κB) signaling in a cell (e.g., an endothelial cell).

BACKGROUND

The vascular endothelium represents a critical interface between bloodand all tissues. Endothelial dysfunction contributes to the developmentof both acute inflammatory disease states, such as endotoxemia andsepsis, and chronic inflammatory disease states, such asatherosclerosis, diabetes, rheumatoid arthritis, and inflammatory boweldisease (Aird, Blood 101:3765-3777, 2003; Aird, Lancet 365:63-78, 2007;Baker et al., Cell Metab. 13:11-22, 2011; Gareus et al., Cell Metab.8:372-383, 2008; Guerci et al., Diabetes Metab. 27:436-477, 2001;Hansson and Libby, Nat. Rev. Immunol. 6:508-519, 2006; Khan et al., Nat.Rev. Rheumatol. 6: 253-261, 2010; Roifman et al., Clin. Gastroenterol.Hepatol. 7:175-182, 2009). In response to inflammatory stimuli, thevascular endothelium expresses a number of adhesion molecules that playkey roles in the recruitment of leukocytes to sites of inflammation (Leyet al., Nat. Rev. Immunol. 7:678-689, 2007; Pober and Sessa, J. Immunol.138:3319-3324, 2007). In particular, vascular cell adhesion molecule 1(VCAM-1), E-selectin, and intercellular adhesion molecule 1 (ICAM-1)mediate early leukocyte attachment and rolling events. An inflammatoryresponse within tissues is subsequently generated after events such asfirm adhesion and transmigration occur (Ley et al., Nat. Rev. Immunol.7:678-689, 2007). Clinical studies have found that the soluble forms ofthese adhesion molecules are increased in patients experiencing vascularinflammatory disease (Shapiro et al., Crit. Care 14:R182, 2010; Xu etal., Int. J. Cardiol. 64:253-258, 1998).

SUMMARY

The present invention is based, at least in part, on the surprisingdiscovery that overexpression of miR-181b in endothelial cells inhibitedTNF-α-induced NF-κB-mediated up-regulation of vascular cell adhesionmolecule-1 (VCAM-1), E-selectin, and intracellular adhesion molecule-1(ICAM-1) expression, inhibited leukocyte adhesion to activatedendothelial cell monolayers, and suppressed TNF-α-induced NF-κB-mediatedVCAM-1 and E-selectin expression in vascular endothelium in vivo. Inview of the discovery that miR-181b inhibits NF-κB signaling, methods oftreating diseases caused or mediated by NF-κB signaling (e.g., vascularinflammatory diseases) and methods of inhibiting NF-κB signaling in acell (e.g., an endothelial cell) are provided.

Accordingly, provided herein are methods of treating or delaying theonset of a vascular inflammatory disease (e.g., lung inflammation (e.g.,acute lung injury, such as sepsis-induced acute lung injury), asthma,atherosclerosis, arthritis, stroke, inflammatory bowel syndrome,cardiovascular disease, myocardial infarction, coronary artery disease,heart failure, ulcerative colitis, Crohn's disease, and peripheralartery disease) including administering to the subject a therapeuticallyeffective amount of a nucleic acid containing all or a part (e.g., 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23nucleotides) of the sequence of miR-181b (SEQ ID NO: 1). In someembodiments of these methods, the vascular inflammatory disease is anacute vascular inflammatory disease or a chronic vascular inflammatorydisease.

In some embodiments of the methods described herein, the nucleic acid isadministered orally, intramuscularly, subcutaneously, arterially,intravenously, or by inhalation. In some embodiments of these methods,the subject is administered one dose or multiple doses (e.g., at leasttwo, three, four, five, six, seven, eight, nine, or ten doses) of thenucleic acid. In some embodiments, the nucleic acid is administeredcontinuously over a treatment period (e.g., at least 5 minutes, 10minutes, 20 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5hours, 6 hours, 10 hours, 18 hours, 24 hours, 48 hours, or 1 week).

In some embodiments of the methods described herein, the administeringresults in a decrease (e.g., a significant (as used herein, the term“significant” means statistically significant) decrease, such as adecrease of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%) in leukocyte adhesion tothe subject's endothelium or a decrease (e.g., a significant decrease,such as a decrease of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%) in leukocyteextravasion of the subject's endothelium as compared to a controlsubject (e.g., a healthy or asymptomatic subject, a subject not havingbeen diagnosed with or not presenting with one or more symptoms of avascular inflammatory disorder, or a subject diagnosed with orpresenting with one or more symptoms of a vascular inflammatorydisorder) not administered the nucleic acid. In some embodiments of themethods described herein, the administering results in a decrease (e.g.,a significant decrease, such as a decrease of at least 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, or 95%) in the expression (protein or nucleic acid) of VCAM-1,E-selectin, or ICAM-1 in the subject's endothelium as compared to acontrol subject (e.g., a healthy or asymptomatic subject, a subject nothaving been diagnosed with or not presenting with one or more symptomsof a vascular inflammatory disorder, or a subject diagnosed with orpresenting with one or more symptoms of a vascular inflammatorydisorder) not administered the nucleic acid.

Also provided are methods of decreasing (e.g., a significant decrease,such as a decrease of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%) nuclearfactor-κB (NF-κB) signaling in an endothelial cell (e.g., an endothelialcell in a subject or an endothelial cell in vitro or ex vivo) includingadministering to the endothelial cell a nucleic acid containing all or apart (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, or 23 nucleotides) of the sequence of miR-181b (SEQ ID NO: 1).In some embodiments, the administering results in a decrease (e.g., asignificant decrease, such as a decrease of at least 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or95%) in the expression (protein or mRNA) of importin-α3 in theendothelial cell as compared to an endothelial cell not administered thenucleic acid or administered a control nucleic acid (e.g., a scramblednon-specific sequence). In some embodiments, the administering resultsin a decrease (e.g., a significant decrease, such as a decrease of atleast 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, or 95%) in the nuclear import of the p65 and/orp50 subunit of NF-κB into the nucleus of the endothelial cell ascompared to an endothelial cell not administered the nucleic acid oradministered a control nucleic acid (e.g., a scrambled non-specificsequence). In some embodiments, the administering results in a decrease(e.g., a significant decrease, such as a decrease of at least 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, or 95%) in the expression (protein or mRNA) of one or more(e.g., one, two, or three) of VCAM-1, E-selectin, or ICAM-1 in theendothelial cell as compared to an endothelial cell not administered thenucleic acid or administered a control nucleic acid (e.g., a scramblednon-specific sequence).

In some embodiments, the endothelial cell is in a subject and thenucleic acid is administered to the subject orally, intramuscularly,subcutaneously, arterially, intravenously, or by inhalation.

In any of the methods described herein, the nucleic acid can contain thesequence of SEQ ID NO: 2 or SEQ ID NO: 3. In any of the methodsdescribed herein, the nucleic acid can be modified or conjugated to oneor more (e.g., two or three) of a polymer (e.g., a polyethylene glycolor a polyalkylene glycol), a peptide (e.g., a RGD peptide or a collagen(e.g., type I telocollagen)), and a polysaccharide (e.g., aβ-1,3-glycan).

As referred to herein, a “complementary nucleic acid sequence” is anucleic acid sequence capable of hybridizing with another nucleic acidsequence comprised of complementary nucleotide base pairs. By“hybridize” is meant pair to form a double-stranded molecule betweencomplementary nucleotide bases (e.g., adenine (A) forms a base pair withthymine (T) (or uracil (U) in the case of RNA), and guanine (G) forms abase pair with cytosine (C)) under suitable conditions of stringency.(See, e.g., Wahl, G. M. and S. L. Berger, Methods Enzymol. 152:399,1987; Kimmel, A. R., Methods Enzymol. 152:507, 1987). For the purposesof the present methods, the nucleic acid need not be complementary tothe entire target sequence (e.g., a sequence within the importin-α3mRNA, such as a sequence within the 3′-UTR of the importin-3α mRNA),only enough of it to provide specific inhibition; for example in someembodiments the sequence is 100% complementary to at least 5-23, 5-15,or 5-10 contiguous nucleotides in the 3′-UTR of a importin-3α mRNA(e.g., site 1 or site 2 in the 3′-UTR of importin-α3 shown in FIG. 8A).Further details are provided below.

As used herein, an “antisense oligonucleotide” refers to a nucleic acidsequence that is complementary to a DNA or RNA sequence, such a sequencepresent in importin-α3 (e.g., a sequence present in the 3′-UTR ofimportin-α3 mRNA, such as site 1 or site 2 in the 3′-UTR of importin-α3mRNA shown in FIG. 8A).

By the term “treating” is meant a reduction in the severity, duration,or frequency of one or more (e.g., two, three, four, five, or six)symptoms of a disease (e.g., a vascular inflammatory disease), anelimination of one or more (e.g., two, three, four, five, or six)symptoms of a disease (e.g., a vascular inflammatory disease), and/or adelay in the onset of one or more (e.g., two, three, four, five, or six)symptoms of a disease (e.g., a vascular inflammatory disease) in asubject (e.g., a subject diagnosed as having a vascular inflammatorydisease or a subject identified as being at risk of developing avascular inflammatory disease). A delay in the onset of one or moresymptoms of a disease (e.g., a vascular inflammatory disease) in asubject administered a nucleic acid containing all or a part of thesequence of SEQ ID NO: 1 may be compared to the development of the samesymptoms in a subject with the same disease (e.g., same vascularinflammatory disease) that is not administered the nucleic acid.

By the term “vascular inflammatory disease” is a disease state thatinvolves at one or more (e.g., one, two, or three) stages (e.g., anearly stage (e.g., before the development of one or more symptoms of avascular inflammatory disease or before diagnosis by a health careprofessional), an intermediate stage (e.g., following the development ofone or more symptoms of a vascular inflammatory disease or followingdiagnosis by a health care professional), or a late stage (e.g.,following the manifestation of one or more severe symptoms of a vascularinflammatory disease that require admission into a health care facility(e.g., a hospital or intensive care unit)) in the pathobiology of thedisease one or more (e.g., one, two, three, or four) of: endothelialcell activation, leukocyte adhesion to the endothelium, leukocyteextravasion of the endothelium, and increased (e.g., a significantincrease, such as by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 70%, 80%, 90%, or 95%) expression (protein or mRNA)of one or more of VCAM-1, E-selectin, or ICAM-1 in the endotheliumcompared to a control subject (e.g., a healthy or asymptomatic subject,a subject not diagnosed with a vascular inflammatory disease, a subjectnot presenting with one or more symptoms of a vascular inflammatorydisease, the same subject prior to the development of one or moresymptoms of a vascular inflammatory disease, the same subject prior todiagnosis with a vascular inflammatory disease, or the same subject atan earlier stage in the vascular inflammatory disease).

By the term “acute vascular inflammatory disease” is meant a vascularinflammatory disease that is typified by an initial response of the bodyto harmful stimuli (e.g., bacterial infection or tissue injury) whichresults in the increased movement of both plasma and leukocytes (e.g.,granulocytes) from the blood into the injured tissue(s).

By the term “chronic vascular inflammatory disease” is meant a vascularinflammatory disease that is characterized by a prolonged period (e.g.,at least 1 week, 2 weeks, one month, two months, three months, fourmonths, five months, six months, 1 year, 2 years, 3 years, 4 years, or 5years) of inflammation in one or more tissues (e.g., two, three, four,of five) in a subject.

By the term “acute lung injury” is meant an inflammatory disease in thelung that results in a decrease in respiratory function. Acute lunginjury is often characterized by one or more (e.g., two, three, or four)of: decreased partial pressure of oxygen in the blood, pulmonary edema,decreased lung compliance, and capillary leakage. Acute lung injury maybe caused by local or systemic inflammation. For example, acute lunginjury may be induced by sepsis (“sepsis-induced acute lung injury.”)

By the term “delaying the onset” is meant reducing (e.g., a significantdecrease, such as by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 70%, 80%, 90%, or 95%) the rate of development orthe development of one or more (e.g., at least two, three, four, orfive) symptoms of disease (e.g., a vascular inflammatory disease) in asubject by administering a therapeutic treatment (e.g., a nucleic acidcontaining all or a part of the sequence of SEQ ID NO: 1) compared to acontrol subject (e.g., a subject not receiving the therapeutic treatmentor the same subject prior to administration of the therapeutictreatment).

By the phrase “a part of the sequence of miR-181b” or “a part of thesequence of SEQ ID NO: 1” is meant 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides within thesequence of SEQ ID NO: 1. For example a part of the sequence of miR-181bmay be between 5 to 23, 5 to 20, 5 to 15, or 5 to 10 contiguousnucleotides within the sequence of SEQ ID NO: 1. The contiguous sequencedoes need to start at the 5′-end of the sequence of SEQ ID NO: 1 and maystart at nucleotide position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, or 18 of SEQ ID NO: 1.

By “therapeutically effective amount” is meant an amount that issufficient to ameliorate or treat one or more (e.g., two, three, four,or five) symptoms of a vascular inflammatory disease in a subject ordelay the onset of one or more (e.g., two, three, four, or fivesymptoms) of a vascular inflammatory disease in a subject.

By “symptom of a vascular inflammatory disease” is a meant a physicalmanifestation of a vascular inflammatory disease that can be assessed ormeasured by a health care professional (e.g., a physician, a nurse, aphysician's assistant, or a laboratory technician). Non-limitingexamples of symptoms of a vascular inflammatory disease include: pain,redness, swelling, vasodilation, increased vascular permeability,diarrhea, nausea, vomiting, abdominal cramps, abdominal pain, blood instool, fever, ulcers, reduced appetite, weight loss, fatigue, eyeinflammation, chest pain or discomfort, shortness of breath, dizziness,increased heart rate, upper body pain, stomach pain, anxiety, sweating,leg cramping, leg numbness or weakness, sores on legs and toes, changein color of legs, hair loss of slower hair growth on feet and legs,shiny skin on legs, weak pulse in legs or feet, erectile dysfunction,persistent cough or wheezing, white or pink blood-tinged phlegm, weightgain from fluid retention, difficulty concentrating, heart palpitations,trouble sleeping caused by shortness of breath, audible whistling orwheezing sound when exhaling, bouts of coughing or wheezing, numbness orweakness in your arms or legs, difficulty speaking or slurred speech,drooping muscles in the face, stiffness in joints, decreased range ofmotion, trouble with seeing in one or both eyes, headache, labored andunusually rapid breathing, low blood pressure, and confusion.

“RNA” is a molecule comprising at least one or more ribonucleotideresidues. A “ribonucleotide” is a nucleotide with a hydroxyl group atthe 2′ position of a beta-D-ribofuranose moiety. The term RNA, as usedherein, includes double-stranded RNA, single-stranded RNA, isolated RNA,such as partially purified RNA, essentially pure RNA, synthetic RNA,recombinantly produced RNA, as well as altered RNA that differs fromnaturally occurring RNA by the addition, deletion, substitution, and/oralteration of one or more nucleotides. Nucleotides of the RNA moleculescan also comprise non-standard nucleotides, such as non-naturallyoccurring nucleotides, chemically-synthesized nucleotides, ordeoxynucleotides.

A “microRNA” (miRNA) is a single-stranded RNA molecule of about 21-23nts in length. In general, miRNAs regulate gene expression. miRNAs areencoded by genes from whose DNA they are transcribed, but miRNAs are nottranslated into protein. Each primary miRNA transcript is processed intoa short stem-loop structure before undergoing further processing into afunctional miRNA. Mature miRNA molecules are partially complementary toone or more messenger RNA (mRNA) molecules, and their main function isto down-regulate gene expression. Exemplary mature miRNAs contain all ora part of the sequence of miR-181b (SEQ ID NO: 1). Exemplary precursormiRNAs contain the sequence of SEQ ID NO: 2 or SEQ ID NO: 3. Someexamples of miRNAs target the 3′-UTR of an importin-α3 mRNA (e.g., site1 and site 2 of the 3′-UTR of an importin-α3 mRNA as shown in FIG. 8A).

By “miR-181b” is meant a nucleic acid molecule containing a sequence ofSEQ ID NO: 1. For example, the term includes precursor miRNA moleculescontaining a sequence of SEQ ID NO: 2 or SEQ ID NO: 3. By “maturemiR-181b” is meant a nucleic acid molecule having the sequence of SEQ IDNO: 1.

As used herein “an interfering RNA” refers to any double stranded orsingle stranded RNA sequence capable, either directly or indirectly, ofinhibiting or down-regulating gene expression by mediating RNAinterference. Interfering RNA includes, but is not limited to, smallinterfering RNA (“siRNA”) and small hairpin RNA (“shRNA”). “RNAinterference” refers to the selective degradation of asequence-compatible messenger RNA transcript (e.g., an importin-α3mRNA). An example of an interfering RNA is a nucleic acid containing allor a part of the sequence of miR-181b (SEQ ID NO: 1). In additionalexamples, an interfering RNA is a nucleic acid containing the sequenceof SEQ ID NO: 2 or SEQ ID NO: 3, or targets the 3′-UTR of an importin-α3mRNA (e.g., site 1 or site 2 of the 3′-UTR of an importin-α3 mRNA asshown in FIG. 8A).

As used herein “an shRNA” (small hairpin RNA) refers to an RNA moleculecomprising an antisense region, a loop portion and a sense region,wherein the sense region has complementary nucleotides that base pairwith the antisense region to form a duplex stem. Followingpost-transcriptional processing, the small hairpin RNA is converted intoa small interfering RNA by a cleavage event mediated by the enzymeDicer, which is a member of the RNase III family. In some examples, anshRNA is a nucleic acid containing all or a part of the sequence ofmiR-181b (SEQ ID NO: 1), SEQ ID NO: 2, or SEQ ID NO: 3. In additionalexamples, an shRNA targets the 3′-UTR of an importin-α3 mRNA (e.g., site1 or site 2 of the 3′-UTR of an importin-α3 mRNA as shown in FIG. 8A).

A “small interfering RNA” or “siRNA” as used herein refers to any smallRNA molecule capable of inhibiting or down-regulating gene expression bymediating RNA interference in a sequence specific manner. The small RNAcan be, for example, about 18 to 21 nucleotides long. An siRNA cancontain all or a part of the sequence of SEQ ID NO: 1. In additionalexamples, an siRNA can target the 3′-UTR of an importin-α3 mRNA (e.g.,site 1 or site 2 of the 3′-UTR of an importin-α3 mRNA as shown in FIG.8A).

A “subject” is a vertebrate, including any member of the class mammalia,including humans, domestic and farm animals, and zoo, sports or petanimals, such as mouse, rabbit, pig, sheep, goat, cattle, and higherprimates. In preferred embodiments, the subject is a human.

As used herein, a “vector” or “expression vector” is a nucleicacid-based delivery vehicle containing regulatory sequences and a geneof interest, which can be used to transfer its contents into a cell. Forexample, the vector may be used to express a nucleic acid containing allor a part of miR-181b (SEQ ID NO: 1) in a cell.

By the term “nuclear factor-κB signaling” or “NF-κB signaling” is meantthe multiple signaling pathways within a cell that result in thetranslocation of NF-κB (e.g., translocation of the p65 and/or p50subunit of NF-κB) into the nucleus and the increased transcription ofone or more (e.g., at least two, three, four, or five) NF-κB-regulatedgenes. In the cytoplasm, NF-κB is complexed with its inhibitor IκB.Upstream signaling pathways activate an IκB kinase (IKK) complex thatresults in IKK-mediated phosphorylation-induced proteasomal degradationof the IκB inhibitor. The degradation of IκB allows NF-κB to translocateto the nucleus and induce transcription by binding to specific genepromoters. As described herein, NF-κB signaling plays a role or has beenimplicated for a role in several disease states (e.g., vascularinflammatory diseases).

By the term “vascular cell adhesion molecule-1” or “VCAM-1” is meant aprotein that is substantially identical (e.g., at least 80%, 85%, 90%,95%, 96%, 97%, 98%, 99%, or 100%) to NCBI Accession No. P19320;NP_001186763.1; NP_542413.1; NP_001069.1; EAW72949.1; AAA61269.1;AAA51917.1; AAA61270.1; AAM96190.1; AAH85003.1; AAH68490.2; orAAH17276.3, or a nucleic acid (e.g., an mRNA) that is substantiallyidentical (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or100%) to NCBI Accession No. NM_001199834.1; NM_001078.3; NM_080682.2;M60335.1; M30257.1; BC085003.1; BC068490.1; BC017276.2; AK223266.1; orX53051.1. Methods for measuring the VCAM-1 protein and mRNA aredescribed herein and are well known in the art.

By the term “E-selectin” is meant a protein that is substantiallyidentical (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or100%) to NCBI Accession No. NP_000441.2 or AAQ67702.1, or a nucleic acid(e.g., an mRNA) that is substantially identical (e.g., at least 80%,85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) to NCBI Accession No.NM_001145667.1 or NM_000450.2. Methods for measuring the E-selectinprotein and mRNA are described herein and are well known in the art.

By the term “intercellular adhesion molecule-1” or “ICAM-1” is meant aprotein that is substantially identical (e.g., at least 80%, 85%, 90%,95%, 96%, 97%, 98%, 99%, or 100%) to NCBI Accession No. CAA41977.1,NP_000192.2, P05362.2, or CAA30051.1, or a nucleic acid (e.g., an mRNA)that is substantially identical (e.g., at least 80%, 85%, 90%, 95%, 96%,97%, 98%, 99%, or 100%) to NCBI Accession No. NG 012083.1, NM_000201.2,J03132.1, or BC015969.2. Methods for measuring the ICAM-1 protein andmRNA are described herein and are well known in the art.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIGS. 1A-1F are data that show that MiR-181b suppresses TNF-α-inducedpro-inflammatory gene expression in human umbilical vein endothelialcells (HUVECs). FIG. 1A is a bar graph showing the expression ofmiR-181b in response to TNF-α (10 ng/ml) treatment in HUVECs. The levelof miR-181b was detected by real-time qPCR. The values representmean±SD. *P<0.01.

FIG. 1B is a bar graph showing that miR-181b is the dominant member ofthe miR-181 family expressed in HUVECs. The levels of miR-181a,miR-181b, and miR-181c were detected by real-time qPCR. The valuesrepresent mean±SD. In FIGS. 1A and 1B, the results represent one out oftwo independently performed experiments with similar outcomes.

FIG. 1C shows the results of Western blot analysis of VCAM-1,E-selectin, and ICAM-1 protein levels in HUVECs transfected with miRNAnegative control (NS-m) or miR-181b mimics (181b-m), miRNA inhibitornegative control (NS-i), or miR-181b inhibitor (181b-i), respectively,after treatment with 10 ng/ml TNF-α for 8 hours (h). Densitometry wasperformed and fold-change of protein expression after normalization toβ-actin expression is shown below each corresponding band. The value forthe NS-m or NS-i group was considered to be 1. Representative images ofat least three experiments are shown.

FIG. 1D is a set of six bar graphs showing the results of real-time qPCRanalysis of VCAM-1 (top row), E-selectin (middle row), and ICAM-1(bottom row) mRNA levels in HUVECs transfected with miRNA negativecontrol (NS-m) or miR-181b mimics (181b-m), miRNA inhibitor negativecontrol (NS-i), or miR-181b inhibitor (181b-i), respectively, aftertreatment with 10 ng/ml TNF-α for the indicated times. The resultsrepresent one out of three independently performed experiments withsimilar outcomes. Values represent mean±SD. #P<0.05 and *P<0.01.

FIG. 1E is a set of six bar graphs showing the results of ELISA analysisof elaborated VCAM-1 (top row), E-selectin (middle row), and ICAM-1(bottom row) protein levels in cell culture medium 16 h after TNF-α (10ng/ml) treatment. HUVECs were transfected as indicated in FIG. 1A.#P<0.05 and *P<0.01. The values represent mean±SD, n=3.

FIG. 1F is a set of six photoimages and two bar graphs showing thatmiR-181b regulates the adhesion of THP-1 cells to TNF-α activatedHUVECs. Photoimages of THP-1 cells adhering to HUVECs transfected withmiRNA negative control (NS-m) (upper and lower far left panels) ormiR-181b mimics (181b-m) (upper and lower middle left panels), miRNAinhibitor negative control (NS-i) (upper and lower middle right panels),or miR-181b inhibitor (181b-i) (upper and lower far right panels),respectively, with (bottom panels) or without (upper panels) 10 ng/mlTNF-α treatment for 4 h are shown. Results represent the mean±SD fromthree independent experiments. *P<0.01, miRNA negative control vs.miR-181b (left bar graph), or miRNA inhibitor negative control vs.miR-181b inhibitor (right bar graph). Bars: 100 μm.

FIGS. 2A-2E is two Western blots and five bar graphs showing thatmiR-181b inhibits pro-inflammatory induction of VCAM-1 expression. FIG.2A is Western blot of VCAM-1 protein levels in HUVECs transfected withmiRNA negative control (NS-m) or miR-181b mimics (181b-m), respectively,and treated with increasing doses of LPS (serotype O26:B6) for 16 h.Densitometry was performed and fold-changes of protein expression afternormalization to β-actin expression are shown in a bar graph in FIG. 2B.Data represent mean±SD from two independent experiments. *P<0.05.

FIG. 2C is three bar graph showing data from an experiment where HUVECswere transfected as in FIG. 2A, and treated with LPS for 6 h. Real-timeqPCR analysis of VCAM-1 (left), E-selectin (center), and ICAM-1 (right)mRNA levels was performed. Values represent mean±SD, n=3. *P<0.05.

FIG. 2D is a bar graph showing that miR-181b does not affect the 3′-UTRactivity of the VCAM-1 gene. Relative luciferase activity of lysatesfrom HUVECs transfected with luciferase VCAM-1 3′-UTR construct in thepresence of miRNA negative control (NS-m) or miR-181b mimics (181b-m) at10 nM or 50 nM, respectively, is shown. Values represent mean±SD, n=3.

FIG. 2E is a Western blot of VCAM-1 in HUVECs transfected with eithermiRNA negative control (NS-m), miR-181b mimics (181b-m), or miR-181amimics (181a-m) at 0.2, 2, 10, or 20 nM concentrations and treated with10 ng/ml TNF-α for 8 h.

FIGS. 3A-E is a Western blot, four bar graphs, and nine photomicrographsthat show that miR-181b represses TNF-α-induced pro-inflammatory geneexpression in vivo. FIG. 3A is a Western blot from an experiment wheremice were intravenously injected with vehicle, miRNA negative control(NS-m), or miR-181b mimics (181b-m). Twenty-four hours later, mice weretreated with or without TNF-α for 4 h, and lungs were harvested forWestern blot analysis of VCAM-1 protein levels. Densitometry wasperformed and fold-change of protein expression after normalization toβ-actin expression was quantified. The value for the vehicle group wasconsidered to be 1. *P<0.05.

FIG. 3B is bar graph showing data from experiments carried out asdescribed in FIG. 3A, where real-time qPCR analysis of VCAM-1 mRNAlevels in the indicated tissues was performed. *P<0.05.

FIG. 3C is a set of nine photomicrographs showing VCAM-1 staining oflung and aorta sections. Mice treated with vehicle in the absence ofTNF-α are shown in the top panels, mice treated with the miRNA negativecontrol (NS-m) and TNF-α are shown in the middle panels, and micetreated with miR-181b mimics (181b-m) are shown in the bottom panels.The middle panels represent an enlargement of the field shown in theleft panels. Mice were treated as in FIG. 3A. Bars: 25 μm.

FIG. 3D is a bar graph showing the quantification of VCAM-1 staining inlung endothelium. *P<0.05.

FIG. 3E is a bar graph showing the quantification of VCAM-1 staining inaorta endothelium. *P<0.05. In FIGS. 3A-E, the vehicle group was 3 mice,the NS-m group was 5 mice, and the 181b-m group was 5 mice. The datarepresent mean±SEM.

FIGS. 4A-C is three bar graphs that show that miR-181b repressesTNF-α-induced pro-inflammatory genes expression in vivo. The data fromreal-time qPCR analysis of E-selectin (bar graph in FIG. 4A) or ICAM-1(bar graph in FIG. 4B) mRNA levels in tissues harvested from miceinjected with vehicle (n=3 mice), miRNA negative control (NS-m) (n=5mice), or miR-181b (181b-m) (n=5 mice) with or without TNF-α treatmentfor 4 h are shown. *P<0.05. The right Y-axis represents the values ofE-selectin expression in liver. Data represent mean±SD.

FIG. 4C is a bar graph showing data from real-time qPCR analysis ofmiR-181b in intima, media plus adventitia of aorta from mice (n=2)injected with miR negative control or miR-181b. *P<0.01. Data representmean±SD.

FIGS. 5A-C is five bar graphs and two Western blots showing thatmiR-181b inhibits activation of NF-κB signaling pathway. FIG. 5A is fourbar graphs of luciferase activity data of reporters containing eitherthe NF-κB concatemer or VCAM-1 promoter in HUVECs transfected with miRNAnegative control (NS-m) or miR-181b mimics (upper left and lower leftgraphs), miRNA inhibitor negative control (NS-i) or miR-181b inhibitor(181b-i) (upper right and lower right graphs) and 12 h after treatmentwith 10 ng/ml of TNF-α. #P<0.05; *P<0.01. Values represent the mean±SD,n=3.

FIG. 5B is a bar graph showing the nuclear p65 staining in HUVECstransfected with miRNA negative control (NS-m) or miR-181b mimics(181b-m).

FIG. 5C is two Western blots showing that the indicated proteins weredetected in cytoplasmic (right blot) or nuclear (left blot) fractionsprepared from HUVECs transfected with miRNA negative control or miR-181bmimics, and treated with 10 ng/ml TNF-α for 1 h. Experiments wereperformed twice. Densitometry was performed and fold-change of p65 andp50 protein expression after normalization is shown below eachcorresponding band.

FIG. 6 is a Western blot of phospho-ERK, total ERK, phospho-p38, totalp38, phospho-JNK, and total JNK in HUVECs transfected with either 10 nMmiRNA negative control or 10 nM miR-181b mimics, and treated with 10ng/ml TNF-α for the indicated times.

FIGS. 7A-F is a Western blot and a set of six bar graphs showing thatmiR-181b reduces importin-α3 expression. FIG. 7A is a Western blot ofimportin-al, importin-α3, and importin-α5 protein levels in cellstransfected with miRNA negative control (NS-m) or miR-181b mimics(181b-m), respectively, in the absence or presence of 10 ng/ml TNF-α.Representative images from at least four experiments are shown.

FIG. 7B is a bar graph showing the normalized luciferase activity dataof a reporter containing the 3′-UTR of importin-α3 mRNA whenco-transfected with increasing amounts of pcDNA3.1 empty vector orpcDNA3.1-miR-181b. Values represent mean±SD, n=3. Results shown are fromone of two independent experiments with similar outcomes. *P<0.01.

FIG. 7C is a bar graph showing the normalized luciferase activity dataof a reporter containing the 3′-UTR of importin-al, importin-α3,importin-α4, or importin-α5, respectively, when co-transfected witheither pcDNA3.1 empty vector or pcDNA3.1-miR-181b. *P<0.01.

FIG. 7D is a bar graph showing the normalized luciferase activity dataof a reporter containing the full-length 3′-UTR of importin-α3,individual rna22 algorithm predicted miR-181b binding sites of the 3′UTRof importin-α3, or mutated 181b binding sites. The reporter wasco-transfected with either pcDNA3.1 empty vector or pcDNA3.1-miR-181b.*P<0.05. In FIGS. 7C and 7D, the values represent mean±SD from threeindependent experiments.

FIG. 7E is two bar graphs showing the data from a miRNP-IP analysis forimportin-α3 or Smad1 mRNA in HUVECs transfected with miRNA negativecontrol (NS-m) or miR-181b mimics (181b-m). Values represent mean±SD oftwo independent experiments. *P<0.01.

FIG. 7F is a bar graph of the luciferase activity data of reporterscontaining the NF-κB concatemer in cells transfected with miRNA negativecontrol (NS-m) or miR-181b mimics (181b-m) in the absence or presence ofimportin-α3 gene lacking its 3′-UTR. Values represent mean±SD, n=3.*P<0.05.

FIGS. 8A-B is a diagram showing eight predicted miR-181b binding sitesand a bar graph showing that miR-181b targets the importin-α3 3′-UTR.FIG. 8A is a diagram of the eight miR-181b binding sites in importin-α33′-UTR that were predicted by rna22. The positions of binding sites areindicated as numbers in parentheses. Lines indicate perfect matches,while colons indicate G:U pairs. Nucleotides marked with dots weremutated. The sequences of miR-181b (SEQ ID NO: 1), 3′UTR site 1 (SEQ IDNO: 120), 3′UTR site 2 (SEQ ID NO: 121), 3′UTR site 3 (SEQ ID NO: 122),3′UTR site 4 (SEQ ID NO: 123), 3′ UTR site 5 (SEQ ID NO: 124), 3′ UTRsite 6 (SEQ ID NO: 125), 3′ UTR site 6 (SEQ ID NO: 126), 3′ UTR site 7(SEQ ID NO: 127), 3′UTR site 8 (SEQ ID NO: 128), mutated 3′UTR site 1(SEQ ID NO: 129), and mutated 3′UTR site 2 (SEQ ID NO: 130) are shown.

FIG. 8B is a bar graph of real-time qPCR data of importin-α3 mRNA levelsin HUVECs transfected with miRNA negative control (NS-m) or miR-181bmimics (181b-m).

FIGS. 9A-D is two bar graphs, a Western blot, and a table showing geneexpression profiling data from HUVECs transfected with miR-181b andbioinformatic analysis of these data. FIG. 9A is a bar graph showing therelative gene expression of 29 TNF-α regulated genes in HUVECstransfected with miRNA negative control (NS-m) or miR-181b mimics(181b-m), as identified by microarray gene chip assay. Expression ispresented as fold-change relative to HUVECs transfected with a miRNAnegative control. The data shown are mean±SD, n=4. *P>0.05.

FIG. 9B is a bar graph showing real-time qPCR analysis of the geneslisted in FIG. 9A. All genes examined were significantly reduced byover-expression of miR-181b (P<0.05).

FIG. 9C is a Western blot of CX3CL-1, PAI-1, COX-2, and VCAM-1 in HUVECstransfected with miRNA negative control (NS-m) or miR-181b mimics(181b-m). Experiments were performed twice.

FIG. 9D is a table showing the results of the gene set enrichmentanalysis.

FIGS. 10A-F is a set of fifteen photomicrographs and five bar graphsthat show that miR-181b reduces endothelial cell activation andleukocyte accumulation in LPS-induced lung inflammation. FIG. 10A is aset of fifteen photomicrographs from an experiment where mice wereintravenously injected with vehicle (top panels), miRNA negative control(NS-m) (middle panels), or miR-181b mimics (181b-m) (bottom panels).Twenty-four hours later, the mice were treated with or without LPS i.p.(40 mg/kg, serotype O26:B6) for 4 h, and lungs were harvested forhistology, then stained for H & E (far left panels), Gr-1 (centerpanels), CD45 (center left panels), or VCAM-1 (center right panels andfar right panels). Bars: 50 μm.

FIG. 10B is a bar graph of lung damage (lung injury score) 4 h after LPSi.p. injection in mice administered vehicle, negative control (NS-m), ormiR-181b mimics (181b-m), as described in FIG. 10A. *P<0.05.

FIG. 10C is a bar graph of quantification data of CD45 positive cells.*P<0.05.

FIG. 10D is a bar graph of quantification data of Gr-1 positive cells.*P<0.05.

FIG. 10E is a bar graph of quantification data of VCAM-1 expression.*P<0.05. In FIGS. 10A-10D, four mice were included in each group, andthe values represent mean±SD.

FIG. 10F is a bar graph of data from an experiment were mice weretreated as in FIG. 10A. Lungs were then harvested and assessed formyeloperoxidase (MPO) activity, and the value of vehicle group was setto 1. The values represent mean±SD, with six mice per group.

FIGS. 11A-D is a set of three photomicrographs and three bar graphs thatshow that miR-181b reduces leukocyte adhesion to the vascularendothelium in LPS-induced lung inflammation.

FIGS. 11A-11B are two bar graphs of real-time qPCR data from anexperiment where mice were treated with TNF-α (2 μg/mouse) i.p., LPSi.p. (40 mg/kg, serotype O26:B6), or saline for 4 h. Aortic intima(endothelium) were isolated for total RNA extraction, followed byreverse transcription, and real-time qPCR analysis. The data forexpression of miR-181b mRNA is shown in FIG. 11B, and the data for theexpression of VCAM-1 mRNA is shown in FIG. 11B. There were 3 to 4 miceper group. The values represent mean±SEM. *P<0.05.

FIG. 11C is a set of three photomicrographs from an experiment wheremice were intravenously injected with vehicle (top), miRNA negativecontrol (NS-m) (middle), or miR-181b mimics (181b-m) (bottom).Twenty-four hours later, the mice were treated with or without LPS i.p.(40 mg/kg, serotype O26:B6) for 4 h, and lungs were harvested for Gr-1staining Bars: 20 μm.

FIG. 11D is a bar graph of quantification data of the number of Gr-1positive cells per mm vessel length. Four mice were included in eachgroup, and the values represent mean±SD. *P<0.05.

DETAILED DESCRIPTION

Provided herein are methods of treating or delaying the onset of adisease induced or mediated by NF-κB signaling (e.g., a vascularinflammatory disease) including administering to the subject atherapeutically effective amount of a nucleic acid containing all or apart of the sequence of mature miR-181b (SEQ ID NO: 1). Also providedare methods of decreasing NF-κB signaling in a cell (e.g., in a subject,in vitro, or ex vivo) including administering to the endothelial cell anucleic acid containing all of a part of the sequence of mature miR-181b(SEQ ID NO: 1).

NF-κB

Induction of VCAM-1, E-selectin, and ICAM-1 in endothelial cells (ECs)is primarily mediated by the activation of the NF-κB pathway. Activationof NF-κB transcription factors have been implicated in manyphysiological and pathological processes (Baker et al., Cell Metab.13:11-22, 2011; Ghosh and Hayden, Nature Rev. Immunol. 8:837-848, 2008;Perkins, Nat. Rev. Mol. Cell. Biol. 8:49-62, 2007). For example, NF-κBhas been implicated for a role in heart failure, ischemia/reperfusion,cardiac hypertrophy, atherosclerosis, multiple sclerosis, musculardystrophy, bone resorption, Alzheimer's disease, incontinentia pigmenti,ectodermal dysplasia, systematic inflammatory response syndrome,inflammatory bowel diseases, neuropathological diseases, heliobacterpylori-associated gastritis, renal diseases, chronic obstructivepulmonary disease, sleep apnea, viral infections (e.g., HIV), skindiseases, gut diseases, sepsis, lupus, aging, diabetes (type I and II),headache, asthma, arthritis, and cancer (Kumar et al., J. Mol. Med.82:434-448, 2004). The present therapies available for the treatment ofthese diseases often result in negative side effects. Thus, newtherapies to treat these diseases (e.g., by inhibiting NF-κB signalingor NF-κB-mediated endothelial cell activation) are desired.

The transcriptional activity of NF-κB can be induced by a variety ofstimuli including the pro-inflammatory cytokines TNF-α and IL-113,growth factors, mitogens, viral or bacterial products (e.g., LPS),T-cell receptor enagement, and stimulation of the CD40 and lymphotoxin-βreceptors (Perkins, Nat. Rev. Mol. Cell Biol. 8:49-62, 2007). In thecanonical NF-κB signaling pathway stimulus-mediated activation of theinhibitor of kappa B (IκB) kinase (IKK) complex leads to IKK rapidlyphosphorylating IκBα at two N-terminal serines, which in turn results inits ubiquitin-induced degradation by the 26S proteasome (Karin andBen-Neriah, Ann. Rev. Immunol. 18:621-663, 2000). This event then leadsto the release of NF-κB heterodimers, which then translocate to thenucleus via importin proteins, and drive a wide range of gene expressionby binding to various κB promoter elements.

In the vascular endothelium, activation of NF-κB leads to the expressionof pro-inflammatory genes, including those encoding cytokines, adhesionmolecules, and chemoattractant proteins that together play criticalroles in all aspects of the inflammatory and immune responses (Blackwelland Christman, Am. J. Respir. Cell Mol. Biol. 17:3-9, 1997; Hajra etal., Proc. Natl. Acad. Sci. U.S.A. 97:9052-9057, 2000; Kempe et al.,Nucleic Acids Res. 33:5308-5319, 2005; Molestina et al., Infect. Immun.68:4282-4288, 2000; Zhou et al., Cell Signal. 19:1238-1248, 2007). Thus,targeting NF-κB-mediated endothelial cell activation holds promise forthe development of new anti-inflammatory therapies.

Vascular Inflammatory Diseases

Endothelial cells perform multiple functions that are critical tovascular homeostasis, including controlling leukocyte trafficking,regulating vessel wall permeability, and maintenance of blood fluidity.The recruitment of leukocytes and extravasation into the blood vesselwall are essential events to normal inflammatory response and relateddisease states. This is a multi-step process through which endothelialcells first express specific adhesion molecules, such as E-selectin andVCAM-1 (Berlin et al., Cell 80:413-422, 1995; Bevilacqua et al., Proc.Natl. Acad. Sci. U.S.A. 84:9238-9242, 1989; Kansas, Blood 88:3259-3287,1996; Ley et al., Nat. Rev. Immunol. 7:678-689, 2007), that facilitateearly attachment to the vascular endothelium. Endothelial cells alsoproduce a variety of C—C and C—X—C chemokines, which act to furtherpromote leukocyte recruitment. After leukocyte trans-migration occurs,invasion of adjacent tissues allows for propagation of the initialinflammatory response.

The discovery that miR-181b can potently inhibit adhesion molecules,chemokines, and other NF-κB-responsive mediators indicate that it canserve to dampen the early and late stages of vascular inflammation.MiR-181b-mediated inhibition is observed for several major physiologicpro-inflammatory mediators, such as TNF-α and LPS (see FIGS. 1, 3, 9,and 10). Functionally, miR-181b impaired leukocyte adhesion to astimulated endothelial cell monolayer in vitro and leukocyteaccumulation in the lungs in vivo (FIGS. 3 and 10). The effect ofmiR-181b was determined to be specific for the NF-κB signaling pathwayas the majority of the TNF-α-inducible genes examined were inhibited bymiR-181b. Moreover, interrogation of the entire set of over 800miR-181b-reduced genes identified by microarray analysis revealed sixbiological signaling pathways associated with NF-κB activation. miR-181bhad no effect on phosphorylation of the MAPK downstream mediators, ERK,p38, and JNK (FIG. 6). These findings show that miR-181b functions as anegative inhibitor of NF-κB signaling events in response topro-inflammatory stimuli in the vascular endothelium.

Accordingly, methods of treating a vascular inflammatory disease areprovided herein. A vascular inflammatory disease is a disease thatinvolves at one or more (e.g., one, two, or three) stages (e.g., anearly stage (e.g., before the development of one or more symptoms of avascular inflammatory disease or before diagnosis by a health careprofessional), an intermediate stage (e.g., following the development ofone or more symptoms of a vascular inflammatory disease or followingdiagnosis by a health care professional), or a late stage (e.g.,following the manifestation of one or more severe symptoms of a vascularinflammatory disease that require admission into a health care facility(e.g., a hospital or an intensive care unit))) in the pathobiology ofthe disease one or more (e.g., one, two, three, or four) of: endothelialcell activation, leukocyte adhesion to the endothelium, leukocyteextravasion of the endothelium, and increased (e.g., a significantincrease, such as by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 70%, 80%, 90%, or 95%) expression (protein or mRNA)in one or more of VCAM-1, E-selectin, or ICAM-1 in the endotheliumcompared to a control subject (e.g., a healthy or asymptomatic subject,a subject not diagnosed with a vascular inflammatory disease, a subjectnot presenting with one or more symptoms of a vascular inflammatorydisease, the same subject prior to the development of one or moresymptoms of a vascular inflammatory disease, the same subject prior todiagnosis with a vascular inflammatory disease, or the same subject atan earlier stage of the vascular inflammatory disease). Non-limitingexamples of vascular inflammatory diseases include: lung inflammation(e.g., acute lung injury, such as sepsis-induced acute lung injury),asthma, atherosclerosis, arthritis, stroke, inflammatory bowel syndrome,cardiovascular disease, myocardial infarction, coronary artery disease,heart failure, ulcerative colitis, Crohn's disease, and peripheralartery disease.

As described above, NF-κB has been implicated for a role in thedevelopment of vascular inflammatory diseases. For example, NF-κBsignaling mediates the up-regulated expression of VCAM-1, E-selectin,and ICAM-1 in endothelial cells, which mediate leukocyte attachment andallow the extravasion of leukocytes to the site of injury. Evidence ofsuch a connection between NF-κB and specific vascular inflammatorydiseases is known. For example, activated NF-κB has been identified insitu in human atherosclerotic plaques, but not in vessels that aredevoid of atherosclerosis (Collins et al., J. Clin. Invest. 107:255-264,2001), NF-κB plays an almost exclusive role in ischemia/reperfusion(Valen et al., J. Am. Coll. Cardiol. 38:307-314, 2001) and in the earlyphase of myocardial infarction (Shimizu et al., Cardiovasc. Res.38:116-124, 1998), activated NF-κB is a common feature in humanrheumatoid arthritis synovium (Marok et al., Arthritis Rheum.39:583-591, 1996; Gilston et al., Biochem. Soc. Trans. 25:518S, 1997;Miyazawa et al., Am. J. Pathol. 152:793-803, 1998) and in various animalmodels of rheumatoid arthritis in rats, collagen-induced arthritis inmice, and streptococcal cell wall induced arthritis in rats (Makarov etal., Arthritis Res. 3:300-206, 2001), and higher NF-κB activation hasbeen reported in colonic biopsy samples, as well as lamina propriamononuclear cells from patients with Crohn's disease (Ellis et al.,Inflamm. Res. 47:440-445, 1998).

A health care professional (e.g., a physician, nurse, a physician'sassistant, or a laboratory technician) may diagnose a subject as havinga vascular inflammatory disease or may monitor the severity, frequency,or duration one or more (e.g., two, three, four, five, or six) symptomsof a vascular inflammatory disease in a subject (e.g., in a subjectreceiving one or more doses of a nucleic acid containing all or a partof the sequence of SEQ ID NO: 1). Such diagnosis may be made usingmethods known in the art (e.g., by the assessment of one or morephysical symptoms of a vascular inflammatory disease known in the art ordiagnostic tests, for e.g., those described in Maksimowic-McKinnon etal., Curr. Opin. Rheumatol. 16:18-24, 2004). Additional laboratory testsfor the diagnosis of specific vascular inflammatory diseases are knownin the art (e.g., C-reactive protein and pro-inflammatory cytokines,such as TNF-α and IL-1). A subject that is diagnosed as having avascular inflammatory disease or is receiving treatment for a vascularinflammatory disease may be admitted to a health care facility (e.g.,hospital, intensive care unit, or an assisted living facility).

Additional Indications

As described above, in addition to its role in vascular inflammatorydiseases, NF-κB has been implicated in other disease states. Forexample, NF-κB signaling has been implicated in obesity,insulin-resistance, diabetes (type I and II), viral infections (e.g.,AIDS), cancer, Alzheimer's disease, muscular dystrophy, bone resorption,ischemia/reperfusion, cardiac hypertrophy, incontinentia pigmenti,ectodermal dysplasia, systematic inflammatory response syndrome,heliobacter pylori-associated gastritis, renal diseases, sleep apnea,skin diseases, lupus, aging, and headache. Transdominant mutants of IκBαthat block NF-κB induction also inhibit de novo HIV-1 infection inT-cells by interfering with viral replication, which indicates thatNF-κB promotes the pathogenesis of HIV-1 in infected cells (Quinto etal., J. Bio. Chem. 274:17567-17572, 1999; Kwon et al., J. Biol. Chem.273:7431-7440, 1998). The ability of NF-κB to suppress apoptosis and toinduce expression of proto-oncogenes, such as c-myc and cyclin Dl, whichdirectly stimulate proliferation, suggest that NF-κB participates inmany aspects of oncogenesis (Pahl, Oncogene 18:6853-6866, 1999; Gutridgeet al., Mol. Cell Biol. 19:5785-5799, 1999). NF-κB also regulates theexpression of various molecules, such as cell adhesion proteins, matrixmetalloproteinases, cyclooxygenase 2, inducible nitric oxide synthase,chemokines, and inflammatory cytokines, all of which promote tumor cellinvasion and angiogenesis (Bharti et al., Biochem. Pharmacol.64:883-888, 2002). In addition, accumulating evidence implicates freeradicals and NF-κB signaling in the destruction of islet β cells anddiabetes disease progression (Ho et al., Proc. Soc. Exp. Biol. Med.222:205-213, 1999). An elevated NF-κB signaling pathway was alsoobserved in the skeletal muscle fibers of patients with polymyositis,dermatomyositis, and Duchenne muscular dystrophy (Monici et al.,Neurology 60:993-997, 2003). NF-κB immunoreactivity was also found inand around early neurological plaque types in Alzheimer's disease(Kaltschmidt et al., Proc. Natl. Acad. Sci. U.S.A. 96:9409-9414, 1999;O'Neill et al., Trends Neurosci. 20:252-258, 1997). Increased activationof NF-κB has also been reported in active multiple sclerosis lesions(Bonetti et al., Am. J. Pathol. 155:1433-1438, 1999; Gveric et al., J.Neuropathol. Exp. Neurol. 57:168-178, 1998). In addition, NF-κBsignaling has been implicated for a mechanistic role in bone resorption(Kumar et al., J. Mol. Med. 82:434-448, 2004).

As NF-κB signaling has been implicated for a role in the development orprogression of these disease states, treatment of these disease statesmay also be performed, in part, by administering a nucleic acidcontaining all or a part of the sequence of mature miR-181b (SEQ ID NO:1), as described for vascular inflammatory diseases below.

MiR-181b

MiRNAs are a class of single-stranded, small non-coding RNAs thattypically bind to the 3′-untranslated region (3′-UTR) of target mRNAsequences, an effect leading to the reduction of protein expressionpredominantly by destabilizing target mRNAs and/or by translationinhibition (Baek et al., Nature 455:64-71, 2008; Bartel, Cell136:215-233, 2009; Guo et al., Nature 466:835-840, 2010;Valencia-Sanchez et al., Genes Dev. 20:515-524, 2006). While over 1,000mature human miRNA sequences are listed in the miRNA registry, only asmall handful have been characterized as functional regulators ofleukocyte or endothelial cell inflammatory responses.

MiR-181b belongs to the miR-181 family which consists of four members:miR-181a, miR-181b, miR-181c, and miR-181d. The biological functions ofthis miRNA family were first identified when miR-181a was recognized asa contributor to hematopoietic lineage commitment and differentiation(Chen et al., Science 303:83-86, 2004; Li et al., Cell 129:147-161,2007). Later studies revealed that increased miR-181a activity inprimary embryonic lymphatic endothelial cells resulted in substantiallyreduced levels of Prox1 mRNA and protein and, consequently, regulatedvascular development and neo-lymphangiogenesis (Kazenwadel et al., Blood116:2395-2401, 2010). MiR-181b was defined as a regulator of the B-cellprimary antibody repertoire based upon its ability to restrict theactivity of activation-induced cytidine deaminase (de Yebenes et al., J.Exp. Med. 205:2199-2206, 2008). Members of the miR-181 family may havenon-redundant functions, as was suggested by the data described hereinand in one study in which miR-181a, but not miR-181c, promoted CD4 andCD8 double-positive T-cell development when ectopically expressed inthymic progenitor cells (Liu et al., PloS One 3:e3592, 2008).

MiR-181b was found to be the dominant miR-181 family member expressed inendothelial cells and was shown to be capable of more potentlysuppressing endothelial cell activation than the next highest expressedfamily member, miR-181a (FIG. 2E). MiR-181b was further discovered totarget the 3′-UTR of importin-α3: a protein that mediates thetranslocation of NF-κB from the cytoplasm into the nucleus.

Importins are a family of proteins involved in nuclear translocation.Using a combination of experimental approaches, includingbioinformatics, 3′-UTR reporter assays, and miRNP-IP, miR-181b was shownto directly target EC-expressed importin-α3 in response to TNF-α.Furthermore, over-expression of importin-α3 (lacking its 3′-UTR)effectively rescued miR-181b-mediated inhibition of NF-κB-inducedactivity (FIG. 7F).

Mature human miR-181b has the sequence of: aacauucauugcugucggugggu (SEQID NO: 1). Mature miR-181b is generated from the processing of aprecursor miRNA molecule, for example, a miR-181b precursor hairpinsequence (hsa-mir-181b-1) encoded on chromosome 1 (Lim et al., Science299:1540, 2003) (CCUGUGCAGAGAUUAUUUUUUAAAAGGUCACAAUCAACAUUCAUUGCUGUCGGUGGGUUGAACUGUGUGGACAAGCUCACUGAACAAUGAAUGCAACUGUGGCCCCGCUU; SEQ ID NO: 2) or amir-181b precursor hairpin sequence (hsa-mir-181b-2) encoded on humanchromosome 9 (Weber, FEBS J. 272:59-73, 2005) (CUGAUGGCUGCACUCAACAUUCAUUGCUGUCGGUGGGUUUGAGUCUGAAUCAACUCACUGAUCAAUGAAUG CAAACUGCGGACCAAACA;SEQ ID NO: 3). In some of the embodiments of the methods describedherein a nucleic acid containing SEQ ID NO: 2 or SEQ ID NO: 3 isadministered to a subject.

The methods provided herein include administering to a subject a nucleicacid that contains all or a part of the sequence of mature miR-181b (SEQID NO: 1). A variety of examples of such nucleic acids are describedbelow. A nucleic acid containing all or a part of the sequence of maturemiR-181b (SEQ ID NO: 1) may include one or more of any of themodifications described herein, without limitation.

Nucleic Acids

Provided herein are methods for treating or delaying the onset of avascular inflammatory disease and methods for decreasing NF-κB signalingin an endothelial cell that include the administration of a nucleic acidthat contains all of a part of the sequence of mature miR-181b (SEQ IDNO: 1). The nucleic acid can be, for example e.g., an antisenseoligonucleotide that contains all or a part of the sequence of maturemiR-181b (SEQ ID NO: 1); in some embodiments, as described in furtherdetail below, the nucleic acid includes different modifications, e.g.,in the sugar backbone, to make it more cell permeable and nucleaseresistant on one hand, and physiologically non-toxic at lowconcentrations on the other. The nucleic acids for use in practicing themethods described herein, that contain all or a part of the sequence ofmature miR-181b (SEQ ID NO: 1), can be those which bind to the 3′-UTR ofimportin-α3 (e.g., bind to a part of site 1 or site 2 of the 3′-UTR ofan importin-α3 mRNA as shown in FIG. 8A) and/or prevent translation ofthe importin-α3 mRNA, such as an interfering RNA, including but notlimited to an shRNA or siRNA.

Inhibitory Nucleic Acids

Nucleic acids useful in the present methods and compositions includemicroRNAs, antisense oligonucleotides, ribozymes, external guidesequence (EGS) oligonucleotides, siRNA compounds, single- ordouble-stranded RNA interference (RNAi) compounds such as siRNAcompounds, modified baseslocked nucleic acids (LNAs), peptide nucleicacids (PNAs), and other oligomeric compounds or oligonucleotide mimeticswhich contain all or a part of the sequence of mature miR-181b (SEQ IDNO: 1) and/or hybridize to at least a portion the 3′UTR of importin-3α(e.g., site 1 or site 2 of the 3′-UTR of an importin-α3 mRNA as shown inFIG. 8A) and modulate its expression. In some embodiments, the nucleicacids include microRNAs, antisense RNA, antisense DNA, chimericantisense oligonucleotides, antisense oligonucleotides comprisingmodified linkages, interference RNA (RNAi), short interfering RNA(siRNA); a micro, interfering RNA (miRNA); a small, temporal RNA(stRNA); or a short, hairpin RNA (shRNA); small RNA-induced geneactivation (RNAa); small activating RNAs (saRNAs), or combinationsthereof. See, for example, WO 10/040112.

In some embodiments, the nucleic acids are 10 to 120, 10 to 110, 10 to100, 10 to 90, 10 to 80, 10 to 70, 10 to 60, 10 to 50, 13 to 50, 13 to30, or 15 to 25 nucleotides in length. One having ordinary skill in theart will appreciate that this embodies oligonucleotides having antisenseportions of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length, or any rangetherewithin. In some embodiments, the oligonucleotides are 15nucleotides in length. In some embodiments, the antisense oroligonucleotide compounds are 12 or 13 to 30 nucleotides in length. Onehaving ordinary skill in the art will appreciate that this embodiesnucleic acids having antisense portions of 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length,or any range therewithin. In some embodiments, the nucleic acid containsat 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or23 continuous nucleotides of SEQ ID NO: 1. In additional embodiments,the nucleic acid contains all or a part (e.g., at least 10, 20, 30, 40,50, 60, 70, 75, 80, 90, or 100 nucleotides of SEQ ID NO: 2 or SEQ ID NO:3).

In some embodiments, the nucleic acids may be designed to target the3′-UTR of importin-α3 as described in the examples (e.g., target site 1and/or site 2 of the 3′-UTR of an importin-α3 mRNA as shown in FIG. 8A).Alternatively or in addition, highly conserved regions within theimportin-α3 mRNA can be targeted, e.g., regions identified by aligningsequences from disparate species such as primate (e.g., human) androdent (e.g., mouse) and looking for regions with high degrees ofidentity. Percent identity can be determined routinely using basic localalignment search tools (BLAST programs) (Altschul et al., J. Mol. Biol.,215:403-410, 1990; Zhang and Madden, Genome Res., 7:649-656, 1997),e.g., using the default parameters.

In some embodiments, the nucleic acids are chimeric oligonucleotidesthat contain two or more chemically distinct regions, each made up of atleast one nucleotide. These oligonucleotides typically contain at leastone region of modified nucleotides that confers one or more beneficialproperties (such as, for example, increased nuclease resistance,increased uptake into cells, increased binding affinity for the target)and a region that is a substrate for enzymes capable of cleaving RNA:DNAor RNA:RNA hybrids. Chimeric nucleic acids used in the methods may beformed as composite structures of two or more oligonucleotides, modifiedoligonucleotides, oligonucleosides, and/or oligonucleotide mimetics asdescribed herein. Such compounds have also been referred to in the artas hybrids or gapmers. Representative U.S. patents that teach thepreparation of such hybrid structures comprise, but are not limited to,U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878;5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and5,700,922, each of which is herein incorporated by reference.

In some embodiments, any of the nucleic acids described herein compriseat least one nucleotide modified at the 2′ position of the sugar, mostpreferably a 2′-O-alkyl, 2′-O-alkyl-O-alkyl, or 2′-fluoro-modifiednucleotide. In other preferred embodiments, RNA modifications include2′-fluoro, 2′-amino, and 2′ O-methyl modifications on the ribose ofpyrimidines, abasic residues, or an inverted base at the 3′ end of theRNA. Such modifications are routinely incorporated into oligonucleotidesand these oligonucleotides have been shown to have a higher Tm (i.e.,higher target binding affinity) than 2′-deoxyoligonucleotides against agiven target.

A number of nucleotide and nucleoside modifications have been shown tomake the oligonucleotide into which they are incorporated more resistantto nuclease digestion than the native oligodeoxynucleotide; thesemodified oligonucleotides survive intact for a longer time thanunmodified oligonucleotides. Specific examples of modifiedoligonucleotides include those comprising modified backbones, forexample, phosphorothioates, phosphotriesters, methyl phosphonates,short-chain alkyl, or cycloalkyl intersugar linkages, or short-chainheteroatomic or heterocyclic intersugar linkages. Most preferred areoligonucleotides with phosphorothioate backbones and those withheteroatom backbones, particularly CH₂—NH—O—CH₂, CH, ˜N(CH₃)˜O˜CH₂(known as a methylene(methylimino) or MMI backbone), CH₂—O—N(CH₃)—CH₂,CH₂—N(CH₃)—N(CH₃)—CH₂ and O—N(CH₃)—CH₂—CH₂ backbones, wherein the nativephosphodiester backbone is represented as O—P—O—CH); amide backbones(see De Mesmaeker et al. Ace. Chem. Res. 28:366-374, 1995); morpholinobackbone structures (see U.S. Pat. No. 5,034,506); and peptide nucleicacid (PNA) backbone (wherein the phosphodiester backbone of theoligonucleotide is replaced with a polyamide backbone, the nucleotidesbeing bound directly or indirectly to the aza nitrogen atoms of thepolyamide backbone, see Nielsen et al., Science 254:1497, 1991).Phosphorus-containing linkages include, but are not limited to,phosphorothioates, chiral phosphorothioates, phosphorodithioates,phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates comprising 3′-alkylene phosphonates and chiralphosphonates, phosphinates, phosphoramidates comprising 3′-aminophosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those having inverted polarity, wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2; see U.S.Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455, 233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563, 253; 5,571,799;5,587,361; and 5,625,050.

Morpholino-based oligomeric compounds are described in Braasch andCorey, Biochemistry 41(14):4503-4510, 2002; Genesis, volume 30, issue 3,2001; Heasman, J., Dev. Biol. 243:209-214, 2002; Nasevicius et al., Nat.Genet. 26:216-220, 2000; Lacerra et al., Proc. Natl. Acad. Sci. U.S.A.97, 9591-9596, 2000; and U.S. Pat. No. 5,034,506. Cyclohexenyl nucleicacid oligonucleotide mimetics are described in Wang et al., J. Am. Chem.Soc. 122:8595-8602, 2000.

Modified oligonucleotide backbones that do not include a phosphorus atomtherein have backbones that are formed by short chain alkyl orcycloalkyl internucleoside linkages, mixed heteroatom and alkyl orcycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These comprisethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S, and CH₂ component parts; see U.S. Pat. Nos. 5,034,506; 5,166,315;5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564;5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307;5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046;5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and5,677,439, each of which is herein incorporated by reference.

One or more substituted sugar moieties can also be included, e.g., oneof the following at the 2′ position: OH, SH, SCH₃, F, OCN, OCH₃OCH₃,OCH₃O(CH₂)nCH₃, O(CH₂)nNH₂ or O(CH₂)nCH₃ where n is from 1 to about 10;C1 to C10 lower alkyl, alkoxyalkoxy, substituted lower alkyl, alkaryl,or aralkyl; Cl; Br; CN; CF₃; OCF₃; O—, S, or N-alkyl; O-, S-, orN-alkenyl; SOCH₃; SO₂CH₃; ONO₂; NO₂; N₃; NH₂; heterocycloalkyl;heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl;an RNA cleaving group; a reporter group; an intercalator; a group forimproving the pharmacokinetic properties of an oligonucleotide; or agroup for improving the pharmacodynamic properties of an oligonucleotideand other substituents having similar properties. A preferredmodification includes 2′-methoxyethoxy[2′-0-CH₂CH₂OCH₃, also known as2′-O-(2-methoxyethyl)] (Martin et al, HeIv. Chim. Acta 78:486, 1995).Other preferred modifications include 2′-methoxy (2′-O—CH₃), 2′-propoxy(2′-OCH₂CH₂CH₃), and 2′-fluoro (2′-F). Similar modifications may also bemade at other positions on the oligonucleotide, particularly the 3′position of the sugar on the 3′ terminal nucleotide and the 5′ positionof 5′ terminal nucleotide. Oligonucleotides may also have sugar mimeticssuch as cyclobutyls in place of the pentofuranosyl group.

Any of the nucleic acids described herein can also include, additionallyor alternatively, nucleobase (often referred to in the art simply as“base”) modifications or substitutions. As used herein, “unmodified” or“natural” nucleobases include adenine (A), guanine (G), thymine (T),cytosine (C), and uracil (U). Modified nucleobases include nucleobasesfound only infrequently or transiently in natural nucleic acids, e.g.,hypoxanthine, 6-methyladenine, 5-Me pyrimidines, particularly5-methylcytosine (also referred to as 5-methyl-2′ deoxycytosine andoften referred to in the art as 5-Me-C), 5-hydroxymethylcytosine (HMC),glycosyl HMC and gentobiosyl HMC, as well as synthetic nucleobases,e.g., 2-aminoadenine, 2-(methylamino)adenine,2-(imidazolylalkyl)adenine, 2-(aminoalklyamino)adenine or otherheterosubstituted alkyladenines, 2-thiouracil, 2-thiothymine,5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N6(6-aminohexyl)adenine and 2,6-diaminopurine. Kornberg, A., DNAReplication, W. H. Freeman & Co., San Francisco, pp 75-77, 1980;Gebeyehu, G., et al. Nucl. Acids Res. 15:4513, 1987). A “universal” baseknown in the art, e.g., inosine, can also be included. 5-Me-Csubstitutions have been shown to increase nucleic acid duplex stabilityby 0.6-1.2° C. (Sanghvi, Y. S., in Crooke, S. T. and Lebleu, B., eds.,Antisense Research and Applications, CRC Press, Boca Raton, pp. 276-278,1993) and are presently preferred base substitutions.

It is not necessary for all positions in a given oligonucleotide to beuniformly modified, and in fact more than one (e.g., two, three, four,or five) of the aforementioned modifications may be incorporated in asingle oligonucleotide or even at within a single nucleoside within anoligonucleotide. Within a given nucleic acid used in the methodsdescribed herein, one or more (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, or 15) of the nucleotides may be modified using anyof the modifications described herein (e.g., sugar, base, orinternucleoside linkage).

In some embodiments, both a sugar and an internucleoside linkage, i.e.,the backbone, of the nucleotide units are replaced with novel groups.The base units are maintained for hybridization with an appropriatenucleic acid target compound (e.g., the 3′-UTR of importin-α3). One sucholigomeric compound, an oligonucleotide mimetic that has been shown tohave excellent hybridization properties, is referred to as a peptidenucleic acid (PNA). In PNA compounds, the sugar-backbone of anoligonucleotide is replaced with an amide containing backbone, forexample, an aminoethylglycine backbone. The nucleobases are retained andare bound directly or indirectly to aza nitrogen atoms of the amideportion of the backbone. Representative U.S. patents that teach thepreparation of PNA compounds comprise, but are not limited to, U.S. Pat.Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference. Further teaching of PNA compounds can befound in Nielsen et al, Science 254:1497-1500, 1991.

Some nucleic acids can also include one or more (e.g., at least 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) nucleobase (often referred toin the art simply as “base”) modifications or substitutions. As usedherein, “unmodified” or “natural” nucleobases comprise the purine basesadenine (A) and guanine (G), and the pyrimidine bases thymine (T),cytosine (C) and uracil (U). Modified nucleobases comprise othersynthetic and natural nucleobases such as: 5-methylcytosine (5-me-C);5-hydroxymethyl cytosine; xanthine; hypoxanthine; 2-aminoadenine;6-methyl and other alkyl derivatives of adenine and guanine; 2-propyland other alkyl derivatives of adenine and guanine; 2-thiouracil;2-thiothymine; 2-thiocytosine; 5-halouracil and cytosine; 5-propynyluracil and cytosine; 6-azo uracil, cytosine, and thymine; 5-uracil(pseudo-uracil); 4-thiouracil; 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl, and other 8-substituted adenines and guanines; 5-halo,particularly 5-bromo, 5-trifluoromethyl, and other 5-substituted uracilsand cytosines; 7-methylquanine; 7-methyladenine; 8-azaguanine;8-azaadenine; 7-deazaguanine; 7-deazaadenine; 3-deazaguanine; and3-deazaadenine.

Further, nucleobases comprise those disclosed in U.S. Pat. No.3,687,808, those disclosed in ‘The Concise Encyclopedia of PolymerScience And Engineering,’ pages 858-859, Kroschwitz, J. I., Ed. JohnWiley & Sons, 1990, those disclosed by Englisch et al., AngewandleChemie, International Edition, volume 30, page 613, 1991, and thosedisclosed by Sanghvi, Y. S., Chapter 15, Antisense Research andApplications, pages 289-302, Crooke, S. T. and Lebleu, B. ea., CRCPress, 1993. Certain of these nucleobases are particularly useful forincreasing the binding affinity of the oligomeric compounds used in themethods described herein. These include 5-substituted pyrimidines,6-azapyrimidines, and N-2, N-6 and 0-6 substituted purines, comprising2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine.5-methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. andLebleu, B., Eds, Antisense Research and Applications, CRC Press, BocaRaton, pp. 276-278, 1993) and are presently preferred basesubstitutions, even more particularly when combined with2′-O-methoxyethyl sugar modifications. Modified nucleobases aredescribed in U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos.4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272;5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540;5,587,469; 5,596,091; 5,614,617; 5,750,692; and 5,681,941, each of whichis herein incorporated by reference.

In some embodiments, any of the nucleic acids described herein arechemically linked to one or more (e.g., two, three, four, five, or six)moieties or conjugates (e.g., a polymer, a peptide, or a polysaccharide)that enhance the activity, cellular distribution, or cellular uptake ofthe oligonucleotide. Such moieties comprise but are not limited to,lipid moieties such as a cholesterol moiety (Letsinger et al., Proc.Natl. Acad. Sci. U.S.A. 86:6553-6556, 1989), cholic acid (Manoharan etal., Bioorg. Med. Chem. Lett. 4:1053-1060, 1994), a thioether, e.g.,hexyl-S— tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci.660:306-309, 1992; Manoharan et al., Bioorg. Med. Chem. Lett.3:2765-2770, 1993), a thiocholesterol (Oberhauser et al., Nucl. AcidsRes. 20:533-538, 1992), an aliphatic chain, e.g., dodecandiol or undecylresidues (Kabanov et al., FEBS Lett. 259:327-330, 1990; Svinarchuk etal., Biochimie 75:49-54, 1993), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett. 36:3651-3654, 1995; Shea et al., Nucl. Acids Res.18:3777-3783, 1990), a polyamine, a polyethylene glycol chain, or apolyalkylene glycol chain (Mancharan et al., Nucleosides & Nucleotides14:969-973, 1995), or adamantane acetic acid (Manoharan et al.,Tetrahedron Lett. 36:3651-3654, 1995), a palmityl moiety (Mishra et al.,Biochim. Biophys. Acta 1264:229-237, 1995), or an octadecylamine orhexylamino-carbonyl-t oxycholesterol moiety (Crooke et al., J.Pharmacol. Exp. Ther. 277:923-937, 1996). See also U.S. Pat. Nos.4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, each of whichis herein incorporated by reference.

These moieties or conjugates can include conjugate groups covalentlybound to functional groups such as primary or secondary hydroxyl groups.Conjugate groups include intercalators, reporter molecules, polyamines,polyamides, polyethylene glycols, polyethers, groups that enhance thepharmacodynamic properties of oligomers, and groups that enhance thepharmacokinetic properties of oligomers. Typical conjugate groupsinclude cholesterols, lipids, phospholipids, biotin, phenazine, folate,phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines,coumarins, and dyes. Groups that enhance the pharmacodynamic properties,in the context of this invention, include groups that improve uptake,enhance resistance to degradation, and/or strengthen sequence-specifichybridization with the target nucleic acid (e.g., the 3′-UTR ofimportin-α3). Groups that enhance the pharmacokinetic properties, in thecontext of this invention, include groups that improve uptake,distribution, metabolism, or excretion of the compounds of the presentinvention. Representative conjugate groups are disclosed in WO93/007883, and U.S. Pat. No. 6,287,860, which are incorporated herein byreference. Conjugate moieties include, but are not limited to, lipidmoieties such as a cholesterol moiety, cholic acid, a thioether, e.g.,hexyl-5-tritylthiol, a thiocholesterol, an aliphatic chain, e.g.,dodecandiol or undecyl residues, a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine, apolyalkylene glycol or a polyethylene glycol chain, adamantane aceticacid, a palmityl moiety, or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety. See, e.g., U.S. Pat. Nos. 4,828,979; 4,948,882;5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717,5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045;5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044;4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263;4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136;5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506;5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723;5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552;5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696;5,599,923; 5,599,928; and 5,688,941.

The nucleic acids useful in the present methods contain all or a part ofthe sequence of mature miR-181b (SEQ ID NO: 1), e.g., hybridizesufficiently well and with sufficient specificity (e.g., complementaryto a sequence present in the 3′-UTR of importin-α3, such as site 1and/or site 2 in the 3′-UTR of an importin-α3 mRNA as shown in FIG. 8A),to give the desired effect. “Complementary” refers to the capacity forpairing, through hydrogen bonding, between two sequences comprisingnaturally or non-naturally occurring bases or analogs thereof. Forexample, if a base at one position of a nucleic acid is capable ofhydrogen bonding with a base at the corresponding position of the target(e.g., a base in the 3′-UTR of importin-α3), then the bases areconsidered to be complementary to each other at that position. 100%complementarity is not required.

In the context of this invention, hybridization means hydrogen bonding,which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogenbonding, between complementary nucleoside or nucleotide bases. Forexample, adenine and thymine are complementary nucleobases which pairthrough the formation of hydrogen bonds. Complementary, as used herein,refers to the capacity for precise pairing between two nucleotides. Thenucleic acids and a sequence present in an importin-α3 mRNA (e.g., asequence present in the 3′-UTR of importin-α3) are complementary to eachother when a sufficient number of corresponding positions in eachmolecule are occupied by nucleotides that can hydrogen bond with eachother. Thus, “specifically hybridizable” and “complementary” are termswhich are used to indicate a sufficient degree of complementarity orprecise pairing such that stable and specific binding occurs between thenucleic acid and the target sequence (e.g., a sequence present in theimportin-α3 mRNA, such as a sequence present in the 3′-UTR ofimportin-α3). For example, if a base at one position of a nucleic acidis capable of hydrogen bonding with a base at the corresponding positionof an importin-α3 mRNA, then the bases are considered to becomplementary to each other at that position.

Although in some embodiments, 100% complementarity is desirable, it isunderstood in the art that a complementary nucleic acid sequence neednot be 100% complementary to that of its target nucleic acid to bespecifically hybridisable. A complementary nucleic acid sequence forpurposes of the present methods is specifically hybridizable whenbinding of the sequence to a importin-α3 mRNA interferes with the normalfunction of the importin-α3 mRNA to cause a loss of activity, and thereis a sufficient degree of complementarity to avoid non-specific bindingof the sequence to non-target mRNA sequences under conditions in whichspecific binding is desired, e.g., under physiological conditions in thecase of in vivo assays or therapeutic treatment, and in the case of invitro assays, under conditions in which the assays are performed undersuitable conditions of stringency. For example, stringent saltconcentration will ordinarily be less than about 750 mM NaCl and 75 mMtrisodium citrate, preferably less than about 500 mM NaCl and 50 mMtrisodium citrate, and more preferably less than about 250 mM NaCl and25 mM trisodium citrate. Low stringency hybridization can be obtained inthe absence of organic solvent, e.g., formamide, while high stringencyhybridization can be obtained in the presence of at least about 35%formamide, and more preferably at least about 50% formamide. Stringenttemperature conditions will ordinarily include temperatures of at leastabout 30° C., more preferably of at least about 37° C., and mostpreferably of at least about 42° C. Varying additional parameters, suchas hybridization time, the concentration of detergent, e.g., sodiumdodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA,are well known to those skilled in the art. Various levels of stringencyare accomplished by combining these various conditions as needed. In apreferred embodiment, hybridization will occur at 30° C. in 750 mM NaCl,75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment,hybridization will occur at 37° C. in 500 mM NaCl, 50 mM trisodiumcitrate, 1% SDS, 35% formamide, and 100 μg/ml denatured salmon sperm DNA(ssDNA). In a most preferred embodiment, hybridization will occur at 42°C. in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and200 μg/ml ssDNA. Useful variations on these conditions will be readilyapparent to those skilled in the art.

For most applications, washing steps that follow hybridization will alsovary in stringency. Wash stringency conditions can be defined by saltconcentration and by temperature. As above, wash stringency can beincreased by decreasing salt concentration or by increasing temperature.For example, stringent salt concentration for the wash steps willpreferably be less than about 30 mM NaCl and 3 mM trisodium citrate, andmost preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate.Stringent temperature conditions for the wash steps will ordinarilyinclude a temperature of at least about 25° C., more preferably of atleast about 42° C., and even more preferably of at least about 68° C. Ina preferred embodiment, wash steps will occur at 25° C. in 30 mM NaCl, 3mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, washsteps will occur at 42° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and0.1% SDS. In a more preferred embodiment, wash steps will occur at 68°C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additionalvariations on these conditions will be readily apparent to those skilledin the art. Hybridization techniques are well known to those skilled inthe art and are described, for example, in Benton and Davis (Science196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci. U.S.A.72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology,Wiley Interscience, New York, 2001); Berger and Kimmel (Guide toMolecular Cloning Techniques, Academic Press, New York, 1987); andSambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, New York.

In general, the nucleic acids useful in the methods described hereinhave at least 80% sequence complementarity to a target region within thetarget nucleic acid, e.g., 90%, 95%, 96%, 97%, 98%, 99%, or 100%sequence complementarity to the target region in an importin-α3 mRNA(e.g., a target region within the 3′-UTR of the importin-α3 mRNA, suchas site 1 and/or site 2 in the 3′-UTR of an importin-α3 mRNA as shown inFIG. 8A). For example, an antisense compound in which 18 of 23nucleobases of the antisense oligonucleotide are complementary, andwould therefore specifically hybridize, to a target region wouldrepresent 90 percent complementarity. Percent complementarity of anucleic acid with a region of a target nucleic acid can be determinedroutinely using basic local alignment search tools (BLAST programs)(Altschul et al., J. Mol. Biol. 215, 403-410, 1990; Zhang and Madden,Genome Res. 7:649-656, 1997). Antisense and other compounds that can beused in the methods that hybridize to an importin-α3 mRNA targetsequence are identified through routine experimentation. In general thenucleic acids must retain specificity for their target, i.e., must notdirectly bind to, or directly significantly affect expression levels of,transcripts other than the intended target.

For further disclosure regarding exemplary nucleic acids that may beused in any one of the methods described herein, please see US2010/0317718 (antisense oligonucleotides); US 2010/0249052(double-stranded ribonucleic acid (dsRNA)); US 2009/0181914 and US2010/0234451 (LNAs); US 2007/0191294 (siRNA analogues); US 2008/0249039(modified siRNA); and WO 10/129746 and WO 10/040112 (inhibitory nucleicacids).

Antisense Nucleic Acids

In some embodiments, the nucleic acids are antisense oligonucleotides.Antisense oligonucleotides are typically designed to block expression ofa DNA or RNA target by binding to the target and halting expression atthe level of transcription, translation, or splicing. Antisenseoligonucleotides of the present invention are complementary nucleic acidsequences designed to hybridize under stringent conditions to acontiguous sequence in an importin-α3 mRNA (e.g., site 1 and/or site 2of the 3′-UTR of an importin-α3 mRNA as shown in FIG. 8A). Thus,oligonucleotides are chosen that are sufficiently complementary to thetarget, i.e., that hybridize sufficiently well and with sufficientspecificity, to give the desired effect.

Modified Bases/Locked Nucleic Acids (LNAs)

In some embodiments, any of the nucleic acids described herein containone or more modified bonds or bases. Modified bases includephosphorothioate, methylphosphonate, peptide nucleic acids, or lockednucleic acid (LNA) molecules. Preferably, the modified nucleotides arelocked nucleic acid molecules, including [alpha]-L-LNAs. LNAs compriseribonucleic acid analogues wherein the ribose ring is “locked” by amethylene bridge between the 2′-oxgygen and the 4′-carbon—i.e.,oligonucleotides containing at least one LNA monomer, that is, one2′-O,4′-C-methylene-β-D-ribofuranosyl nucleotide. LNA bases formstandard Watson-Crick base pairs but the locked configuration increasesthe rate and stability of the base pairing reaction (Jepsen et al.,Oligonucleotides 14:130-146, 2004). LNAs also have increased affinity tobase pair with RNA as compared to DNA. These properties render LNAsespecially useful as probes for fluorescence in situ hybridization(FISH) and comparative genomic hybridization, and as antisenseoligonucleotides to target mRNAs or other RNAs.

The LNA molecules can include molecules comprising 10-120, 10-110,10-100, 10-90, 10-80, 10-70, 10-60, 10-50, 10-40, 10-30, e.g., 12-24,e.g., 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, or 30 nucleotides in each strand, wherein one of the strands issubstantially identical, e.g., at least 80% (or more, e.g., 85%, 90%,95%, 96%, 97%, 98%, 99%, or 100%) identical, e.g., having 3, 2, 1, or 0mismatched nucleotide(s), to a target sequence (e.g., a sequence presentin an importin-α3 mRNA, such as a sequence present in the 3′-UTR of animportin-α3 mRNA). The LNA molecules can be chemically synthesized usingmethods known in the art. The LNA molecules can be designed using anymethod known in the art; a number of algorithms are known, and arecommercially available (e.g., on the internet, for example at the Exiqonwebsite). See, e.g., You et al., Nuc. Acids. Res. 34:e60, 2006; McTigueet al., Biochemistry 43:5388-405, 2004; and Levin et al., Nuc. Acids.Res. 34:e142, 2006. For example, “gene walk” methods, similar to thoseused to design antisense oligonucleotides, can be used to optimize theinhibitory activity of the LNA (or any other nucleic acid describedherein); for example, a series of oligonucleotides of 10-30 nucleotidesspanning the length of a target importin-α3 sequence can be prepared,followed by testing for activity. Optionally, gaps, e.g., of 5-10nucleotides or more, can be left between the LNAs to reduce the numberof oligonucleotides synthesized and tested. The GC content is preferablybetween about 30-60%. General guidelines for designing LNAs are known inthe art; for example, LNA sequences will bind very tightly to other LNAsequences, so it is preferable to avoid significant complementaritywithin an LNA. Contiguous runs of three or more Gs or Cs, or more thanfour LNA residues, should be avoided where possible (for example, it maynot be possible with very short (e.g., about 9-10 nt) oligonucleotides).In some embodiments, the LNAs are xylo-LNAs. For additional informationregarding LNAs see U.S. Pat. Nos. 6,268,490; 6,734,291; 6,770,748;6,794,499; 7,034,133; 7,053,207; 7,060,809; 7,084,125; and 7,572,582;and U.S. Patent Application Publication Nos. 20100267018; 20100261175;and 20100035968; Koshkin et al. Tetrahedron 54:3607-3630, 1998; Obika etal., Tetrahedron Lett. 39:5401-5404, 1998; Jepsen et al.,Oligonucleotides 14:130-146, 2004; Kauppinen et al., Drug Disc. Today2(3):287-290, 2005; and Ponting et al., Cell 136(4):629-641, 2009, andreferences cited therein.

siRNA/shRNA

In some embodiments, the nucleic acid can be an interfering RNA,including but not limited to a small interfering RNA (“siRNA”) or asmall hairpin RNA (“shRNA”). Methods for constructing interfering RNAsare well known in the art. For example, the interfering RNA can beassembled from two separate oligonucleotides, where one strand is thesense strand and the other is the antisense strand, wherein theantisense and sense strands are self-complementary (i.e., each strandcomprises nucleotide sequence that is complementary to nucleotidesequence in the other strand; such as where the antisense strand andsense strand form a duplex or double stranded structure); the antisensestrand comprises nucleotide sequence that is complementary to anucleotide sequence in a target nucleic acid molecule (e.g.,complementary to a sequence present in an importin-α3 mRNA, such as site1 and/or site 2 in the 3′-UTR of an importin-α3 mRNA as shown in FIG.8A) or a portion thereof and the sense strand comprises nucleotidesequence corresponding to the target nucleic acid sequence (e.g., asequence present in an importin-α3 mRNA, such as site 1 and/or site 2 inthe 3′-UTR of an importin-α3 mRNA as shown in FIG. 8A) or a portionthereof. Alternatively, interfering RNA is assembled from a singleoligonucleotide, where the self-complementary sense and antisenseregions are linked by means of nucleic acid based or non-nucleicacid-based linker(s). The interfering RNA can be a polynucleotide with aduplex, asymmetric duplex, hairpin, or asymmetric hairpin secondarystructure, having self-complementary sense and antisense regions,wherein the antisense region comprises a nucleotide sequence that iscomplementary to nucleotide sequence in a separate target nucleic acidmolecule or a portion thereof and the sense region having nucleotidesequence corresponding to the target nucleic acid sequence or a portionthereof. The interfering RNA can be a circular single-strandedpolynucleotide having two or more loop structures and a stem comprisingself-complementary sense and antisense regions, wherein the antisenseregion comprises nucleotide sequence that is complementary to nucleotidesequence in a target nucleic acid molecule or a portion thereof and thesense region having nucleotide sequence corresponding to the targetnucleic acid sequence or a portion thereof, and wherein the circularpolynucleotide can be processed either in vivo or in vitro to generatean active siRNA molecule capable of mediating RNA interference.

In some embodiments, the interfering RNA coding region encodes aself-complementary RNA molecule having a sense region, an antisenseregion and a loop region. Such an RNA molecule when expressed desirablyforms a “hairpin” structure, and is referred to herein as an “shRNA.”The loop region is generally between about 2 and about 10 nucleotides inlength. In some embodiments, the loop region is from about 6 to about 9nucleotides in length. In some embodiments, the sense region and theantisense region are between about 15 and about 20 nucleotides inlength. Following post-transcriptional processing, the small hairpin RNAis converted into a siRNA by a cleavage event mediated by the enzymeDicer, which is a member of the RNase III family. The siRNA is thencapable of inhibiting the expression of a gene with which it shareshomology. For details, see Brummelkamp et al., Science 296:550-553,2002; Lee et al, Nature Biotechnol. 20:500-505, 2002; Miyagishi andTaira, Nature Biotechnol. 20:497-500, 2002; Paddison et al., Genes &Dev. 16:948-958, 2002; Paul, Nature Biotechnol. 20:505-508, 2002; Sui,Proc. Natl. Acad. Soc. U.S.A. 99(6):5515-5520, 2002; Yu et al., Proc.Natl. Acad. Sci. U.S.A. 99:6047-6052, 2002.

The target RNA cleavage reaction guided by siRNAs is highly sequencespecific. In general, siRNA containing a nucleotide sequences identicalto a portion of the target nucleic acid are preferred for inhibition.However, 100% sequence identity between the siRNA and the target gene isnot required to practice the present invention. Thus the providedmethods have the advantage of being able to tolerate sequence variationsthat might be expected due to genetic mutation, strain polymorphism, orevolutionary divergence. For example, siRNA sequences with insertions,deletions, and single point mutations relative to the target sequencehave also been found to be effective for inhibition. Alternatively,siRNA sequences with nucleotide analog substitutions or insertions canbe effective for inhibition. In general the siRNAs must retainspecificity for their target, i.e., must not directly bind to, ordirectly significantly affect expression levels of, transcripts otherthan the intended target (e.g., an importin-α3 mRNA).

Ribozymes

Trans-cleaving enzymatic nucleic acid molecules can also be used; theyhave shown promise as therapeutic agents for human disease (Usman &McSwiggen, Ann. Rep. Med. Chem. 30:285-294, 1995; Christoffersen andMarr, J. Med. Chem. 38:2023-2037, 1995). Enzymatic nucleic acidmolecules can be designed to cleave an importin-α3 mRNA within thebackground of cellular RNA. Such a cleavage event renders theimportin-α3 mRNA non-functional.

In general, enzymatic nucleic acids with RNA cleaving activity act byfirst binding to a target RNA. Such binding occurs through the targetbinding portion of an enzymatic nucleic acid which is held in closeproximity to an enzymatic portion of the molecule that acts to cleavethe target RNA. Thus, the enzymatic nucleic acid first recognizes andthen binds a target RNA through complementary base pairing, and oncebound to the correct site, acts enzymatically to cut the target RNA.Strategic cleavage of such a target RNA will destroy its ability todirect synthesis of an encoded protein (an importin-α3 protein). Afteran enzymatic nucleic acid has bound and cleaved its RNA target, it isreleased from that RNA to search for another target and can repeatedlybind and cleave new targets.

Several approaches such as in vitro selection (evolution) strategies(Orgel, Proc. R. Soc. London, B 205:435, 1979) have been used to evolvenew nucleic acid catalysts capable of catalyzing a variety of reactions,such as cleavage and ligation of phosphodiester linkages and amidelinkages (Joyce, Gene, 82:83-87, 1989; Beaudry et al., Science257:635-641, 1992; Joyce, Scientific American 267:90-97, 1992; Breakeret al., TIBTECH 12:268, 1994; Bartel et al., Science 261:1411-1418,1993; Szostak, TIBS 17:89-93, 1993; Kumar et al., FASEB J., 9:1183,1995; Breaker, Curr. Op. Biotech. 1:442, 1996).

Making and Using the Nucleic Acids

The nucleic acids used to practice the methods described herein, whetherRNA, cDNA, genomic DNA, vectors, viruses or hybrids thereof, can beisolated from a variety of sources, genetically engineered, amplified,and/or expressed/generated recombinantly. Recombinant nucleic acidsequences can be individually isolated or cloned and tested for adesired activity. Any recombinant expression system can be used,including, for e.g., in vitro bacterial, fungal, mammalian, yeast,insect, or plant cell expression systems. Nucleic acid sequences thatcan be used in any of the methods described herein can be inserted intodelivery vectors and expressed from transcription units within thevectors. The recombinant vectors can be DNA plasmids or viral vectors.Generation of the vector construct can be accomplished using anysuitable genetic engineering techniques well known in the art,including, without limitation, the standard techniques of PCR,oligonucleotide synthesis, restriction endonuclease digestion, ligation,transformation, plasmid purification, and DNA sequencing, for example,as described in Sambrook et al., Molecular Cloning: A LaboratoryManual., 1989; Coffin et al., Retroviruses, 1997; and “RNA Viruses: APractical Approach” (Alan J. Cann, Ed., Oxford University Press, 2000).As will be apparent to one of ordinary skill in the art, a variety ofsuitable vectors are available for transferring nucleic acids (e.g., anucleic acid containing all or a part of SEQ ID NO: 1) into cells. Theselection of an appropriate vector to deliver nucleic acids andoptimization of the conditions for insertion of the selected expressionvector into the cell, are within the scope of one of ordinary skill inthe art without the need for undue experimentation. Viral vectorscomprise a nucleotide sequence having sequences for the production ofrecombinant virus in a packaging cell. Viral vectors expressing nucleicacids (e.g., a sequence containing all or a part of SEQ ID NO: 1) can beconstructed based on viral backbones including, but not limited to, aretrovirus, lentivirus, adenovirus, adeno-associated virus, pox virus,or alphavirus. The recombinant vectors capable of expressing a nucleicacid (e.g., a nucleic acid containing all or part of SEQ ID NO: 1) canbe delivered as described herein, and persist in target cells (e.g.,stable transformants).

Nucleic acid sequences containing all or a part of SEQ ID NO: 1 can besynthesized in vitro by well-known chemical synthesis techniques, asdescribed in, e.g., Adams, J. Am. Chem. Soc. 105:661, 1983; Belousov,Nucleic Acids Res. 25:3440-3444, 1997; Frenkel, Free Radic. Biol. Med.19:373-380, 1995; Blommers, Biochemistry 33:7886-7896, 1994; Narang,Meth. Enzymol. 68:90, 1979; Brown, Meth. Enzymol. 68:109, 1979;Beaucage, Tetra. Lett. 22:1859, 1981; and U.S. Pat. No. 4,458,066.

Nucleic acid sequences containing all or a part of SEQ ID NO: 1 can bestabilized against nucleolytic degradation such as by the incorporationof a modification, e.g., a nucleotide modification. For example, nucleicacid sequences (e.g., nucleic acids including all or a part of SEQ IDNO: 1) can include a phosphorothioate at least the first, second, orthird internucleotide linkage at the 5′ or 3′ end of the nucleotidesequence. As another example, the nucleic acid sequence can include a2′-modified nucleotide, e.g., a 2′-deoxy, 2′-deoxy-2′-fluoro,2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl (2′-O-AP),2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl(2′-O-DMAP), 2′-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or2′-O—N-methylacetamido (2′-O—NMA). As another example, the nucleic acidsequence can include at least one 2′-O-methyl-modified nucleotide, andin some embodiments, all of the nucleotides include a 2′-O-methylmodification. In some embodiments, the nucleic acids are “locked,” i.e.,comprise nucleic acid analogues in which the ribose ring is “locked” bya methylene bridge connecting the 2′-O atom and the 4′-C atom (see,e.g., Kaupinnen et al., Drug Disc. Today 2(3):287-290, 2005; Koshkin etal., J. Am. Chem. Soc. 120(50):13252-13253, 1998). For additionalmodifications see US 2010/0004320, US 2009/0298916, and US 2009/0143326.

Techniques for the manipulation of nucleic acids used to practice thisinvention, such as, e.g., subcloning, labeling probes (e.g.,random-primer labeling using Klenow polymerase, nick translation, andamplification), sequencing, hybridization, and the like are welldescribed in the scientific and patent literature, see, e.g., Sambrooket al., Molecular Cloning; A Laboratory Manual 3d ed., 2001; CurrentProtocols in Molecular Biology, Ausubel et al., Eds. (John Wiley & Sons,Inc., New York, 2010); Kriegler, Gene Transfer and Expression: ALaboratory Manual, 1990; Laboratory Techniques in Biochemistry andMolecular Biology: Hybridization with Nucleic Acid Probes, Part I.Theory and Nucleic Acid Preparation, Tijssen, Ed., Elsevier, N.Y., 1993.

Pharmaceutical Compositions

The methods described herein can include the administration ofpharmaceutical compositions and formulations comprising the nucleic acidsequences described herein (e.g., nucleic acids containing all or a partof the sequence of SEQ ID NO: 1).

In some embodiments, the compositions are formulated with apharmaceutically acceptable carrier. The pharmaceutical compositions andformulations can be administered parenterally, intramuscularly,subcutaneously, arterially, intravenously, topically, orally, or bylocal administration, such as by aerosol or transdermally. Thepharmaceutical compositions can be formulated in any way and can beadministered in a variety of unit dosage forms depending upon thecondition or disease and the degree of illness, the general medicalcondition of each patient, the resulting preferred method ofadministration and the like. Details on techniques for formulation andadministration of pharmaceuticals are well described in the scientificand patent literature, see, e.g., Remington: The Science and Practice ofPharmacy, 21st ed., 2005.

The nucleic acids can be administered alone or as a component of apharmaceutical formulation (composition). The compounds may beformulated for administration, in any convenient way for use in human orveterinary medicine. Wetting agents, emulsifiers, and lubricants, suchas sodium lauryl sulfate and magnesium stearate, as well as coloringagents, release agents, coating agents, sweetening, flavoring, perfumingagents, preservatives, and antioxidants can also be present in thecompositions. Formulations of the compositions that may be used in themethods described herein include those suitable for intradermal,inhalation, intramuscular, subcutaneous, arterial, intravenous,oral/nasal, topical, parenteral, rectal, and/or intravaginaladministration. The formulations may conveniently be presented in unitdosage form and may be prepared by any methods well known in the art ofpharmacy. The amount of active ingredient (e.g., a nucleic acid sequencedescribed herein) which can be combined with a carrier material toproduce a single dosage form will vary depending upon the host beingtreated, the particular mode of administration, e.g., intravenous orinhalation. The amount of active ingredient which can be combined with acarrier material to produce a single dosage form will generally be thatamount of the compound which produces a therapeutic effect

Pharmaceutical formulations of this invention can be prepared accordingto any method known to the art for the manufacture of pharmaceuticals.Such drugs can contain sweetening agents, flavoring agents, coloringagents, and preserving agents. A formulation can be admixed withnontoxic pharmaceutically acceptable excipients which are suitable formanufacture. Formulations may comprise one or more diluents,emulsifiers, preservatives, buffers, excipients, etc., and may beprovided in such forms as liquids, powders, emulsions, lyophilizedpowders, sprays, creams, lotions, controlled release formulations,tablets, pills, gels, on patches, in implants, etc.

Pharmaceutical formulations for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art inappropriate and suitable dosages. Such carriers enable thepharmaceuticals to be formulated in unit dosage forms as tablets, pills,powder, dragees, capsules, liquids, lozenges, gels, syrups, slurries,suspensions, etc., suitable for ingestion by the patient. Pharmaceuticalpreparations for oral use can be formulated as a solid excipient,optionally grinding a resulting mixture, and processing the mixture ofgranules, after adding suitable additional compounds, if desired, toobtain tablets or dragee cores. Suitable solid excipients arecarbohydrate or protein fillers include, e.g., sugars, includinglactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice,potato, or other plants; cellulose such as methyl cellulose,hydroxypropylmethyl-cellulose, or sodium carboxy-methylcellulose; andgums including arabic and tragacanth; and proteins, e.g., gelatin andcollagen. Disintegrating or solubilizing agents may be added, such asthe cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a saltthereof, such as sodium alginate. Push-fit capsules can contain activeagents mixed with a filler or binders such as lactose or starches,lubricants such as talc or magnesium stearate, and, optionally,stabilizers. In soft capsules, the active agents can be dissolved orsuspended in suitable liquids, such as fatty oils, liquid paraffin, orliquid polyethylene glycol with or without stabilizers.

Aqueous suspensions can contain an active agent (e.g., nucleic acidsequences containing all or a part of SEQ ID NO: 1) in admixture withexcipients suitable for the manufacture of aqueous suspensions, e.g.,for aqueous intradermal injections. Such excipients include a suspendingagent, such as sodium carboxymethylcellulose, methylcellulose,hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gumtragacanth and gum acacia, and dispersing or wetting agents such as anaturally-occurring phosphatide (e.g., lecithin), a condensation productof an alkylene oxide with a fatty acid (e.g., polyoxyethylene stearate),a condensation product of ethylene oxide with a long chain aliphaticalcohol (e.g., heptadecaethylene oxycetanol), a condensation product ofethylene oxide with a partial ester derived from a fatty acid and ahexitol (e.g., polyoxyethylene sorbitol mono-oleate), or a condensationproduct of ethylene oxide with a partial ester derived from fatty acidand a hexitol anhydride (e.g., polyoxyethylene sorbitan mono-oleate).The aqueous suspension can also contain one or more preservatives suchas ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, oneor more flavoring agents, and one or more sweetening agents, such assucrose, aspartame, or saccharin. Formulations can be adjusted forosmolarity.

In some embodiments, oil-based pharmaceuticals are used foradministration of nucleic acid sequences (e.g., nucleic acid sequencescontaining all or a part of SEQ ID NO: 1). Oil-based suspensions can beformulated by suspending an active agent in a vegetable oil, such asarachis oil, olive oil, sesame oil, or coconut oil, or in a mineral oilsuch as liquid paraffin; or a mixture of these. See e.g., U.S. Pat. No.5,716,928, describing using essential oils or essential oil componentsfor increasing bioavailability and reducing inter- and intra-individualvariability of orally administered hydrophobic pharmaceutical compounds(see also U.S. Pat. No. 5,858,401). The oil suspensions can contain athickening agent, such as beeswax, hard paraffin, or cetyl alcohol.Sweetening agents can be added to provide a palatable oral preparation,such as glycerol, sorbitol, or sucrose. These formulations can bepreserved by the addition of an antioxidant such as ascorbic acid. As anexample of an injectable oil vehicle, see Minto, J. Pharmacol. Exp.Ther. 281:93-102, 1997.

Pharmaceutical formulations can also be in the form of oil-in-wateremulsions. The oily phase can be a vegetable oil or a mineral oil,described herein, or a mixture of these. Suitable emulsifying agentsinclude naturally-occurring gums, such as gum acacia and gum tragacanth,naturally-occurring phosphatides, such as soybean lecithin, esters, orpartial esters derived from fatty acids and hexitol anhydrides, such assorbitan mono-oleate, and condensation products of these partial esterswith ethylene oxide, such as polyoxyethylene sorbitan mono-oleate. Theemulsion can also contain sweetening agents and flavoring agents, as inthe formulation of syrups and elixirs. Such formulations can alsocontain a demulcent, a preservative, or a coloring agent. In alternativeembodiments, the injectable oil-in-water emulsions that may be used inthe methods described herein comprise a paraffin oil, a sorbitanmonooleate, an ethoxylated sorbitan monooleate, and/or an ethoxylatedsorbitan trioleate.

The pharmaceutical compounds can also be administered by in intranasal,intraocular, and intravaginal routes including suppositories,insufflation, powders, and aerosol formulations (for examples ofinhalants, see e.g., Rohatagi, J. Clin. Pharmacol. 35:1187-1193, 1995;Tjwa, Ann. Allergy Asthma Immunol. 75:107-111, 1995). Suppositoriesformulations can be prepared by mixing the drug with a suitablenon-irritating excipient which is solid at ordinary temperatures butliquid at body temperatures and will therefore melt in the body torelease the drug. Such materials are cocoa butter and polyethyleneglycols.

In some embodiments, the pharmaceutical compounds can be deliveredtransdermally, by a topical route, formulated as applicator sticks,solutions, suspensions, emulsions, gels, creams, ointments, pastes,jellies, paints, powders, and aerosols. In some embodiments, thepharmaceutical compounds can also be delivered as microspheres for slowrelease in the body. For example, microspheres can be administered viaintradermal injection of drug which slowly release subcutaneously; seeRao, J. Biomater Sci. Polym. Ed. 7:623-645, 1995; as biodegradable andinjectable gel formulations, see, e.g., Gao, Pharm. Res. 12:857-863,1995; or as microspheres for oral administration, see, e.g., Eyles, J.Pharm. Pharmacol. 49:669-674, 1997.

In some embodiments, the pharmaceutical compounds can be parenterallyadministered, such as by intravenous (IV) administration oradministration into a body cavity or lumen of an organ (e.g., into thelung). These formulations can comprise a solution of active agentdissolved in a pharmaceutically acceptable carrier. Acceptable vehiclesand solvents that can be employed are water and Ringer's solution, anisotonic sodium chloride. In addition, sterile fixed oils can beemployed as a solvent or suspending medium. For this purpose any blandfixed oil can be employed including synthetic mono- or diglycerides. Inaddition, fatty acids such as oleic acid can likewise be used in thepreparation of injectables. These solutions are sterile and generallyfree of undesirable matter. These formulations may be sterilized byconventional, well-known sterilization techniques. The formulations maycontain pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions such as pH adjusting and bufferingagents, toxicity adjusting agents, e.g., sodium acetate, sodiumchloride, potassium chloride, calcium chloride, sodium lactate, and thelike. The concentration of active agent in these formulations can varywidely, and will be selected primarily based on fluid volumes,viscosities, body weight, and the like, in accordance with theparticular mode of administration selected and the patient's needs. ForIV administration, the formulation can be a sterile injectablepreparation, such as a sterile injectable aqueous or oleaginoussuspension. This suspension can be formulated using those suitabledispersing or wetting agents and suspending agents. The sterileinjectable preparation can also be a suspension in a nontoxicparenterally-acceptable diluent or solvent, such as a solution of1,3-butanediol. The administration can be by bolus or continuousinfusion (e.g., substantially uninterrupted introduction into a bloodvessel for a specified period of time).

In some embodiments, the pharmaceutical compounds and formulations canbe lyophilized. Stable lyophilized formulations comprising a nucleicacid can be made by lyophilizing a solution comprising a pharmaceuticaldescribed herein (e.g., containing a nucleic acid containing all or apart of SEQ ID NO: 1) and a bulking agent, e.g., mannitol, trehalose,raffinose, and sucrose, or mixtures thereof. A process for preparing astable lyophilized formulation can include lyophilizing a solution about2.5 mg/mL protein, about 15 mg/mL sucrose, about 19 mg/mL NaCl, and asodium citrate buffer having a pH greater than 5.5, but less than 6.5.See, e.g., US 2004/0028670. The compositions and formulations can bedelivered by the use of liposomes. By using liposomes, particularlywhere the liposome surface carries ligands specific for target cells, orare otherwise preferentially directed to a specific organ, one can focusthe delivery of the active agent into target cells in vivo. See, e.g.,U.S. Pat. Nos. 6,063,400 and 6,007,839; Al-Muhammed, J. Microencapsul.13:293-306, 1996; Chonn, Curr. Opin. Biotechnol. 6:698-708, 1995; Ostro,Am. J. Hosp. Pharm. 46:1576-1587, 1989. As used in the presentinvention, the term “liposome” means a vesicle composed of amphiphiliclipids arranged in a bilayer or bilayers. Liposomes are unilamellar ormultilamellar vesicles that have a membrane formed from a lipophilicmaterial and an aqueous interior that contains the composition to bedelivered. Cationic liposomes are positively-charged liposomes that arebelieved to interact with negatively-charged DNA molecules to form astable complex. Liposomes that are pH-sensitive or negatively-chargedare believed to entrap DNA rather than complex with it. Both cationicand noncationic liposomes have been used to deliver DNA to cells.

Liposomes can also include “sterically-stabilized” liposomes, i.e.,liposomes comprising one or more specialized lipids. When incorporatedinto liposomes, these specialized lipids result in liposomes withenhanced circulation lifetimes relative to liposomes lacking suchspecialized lipids. Examples of sterically-stabilized liposomes arethose in which part of the vesicle-forming lipid portion of the liposomecomprises one or more glycolipids or is derivatized with one or morehydrophilic polymers, such as a polyethylene glycol (PEG) moiety.Liposomes and their uses are further described in U.S. Pat. No.6,287,860.

Methods of Treating a Vascular Inflammatory Disease

Provided herein are methods of treating or delaying the onset of avascular inflammatory disease in a subject including administering tothe subject a nucleic acid containing all or a part of the sequence ofmature miR-181b (SEQ ID NO: 1) (e.g., a sequence containing SEQ ID NO: 2or SEQ ID NO: 3).

Subject that may be treated by the methods of the present invention mayhave been previously diagnosed as having a vascular inflammatorydisease, may present with one or more (e.g., two, three, four, or five)symptoms of a vascular inflammatory disease, may have an increased riskof developing a vascular inflammatory disease, may be admitted in amedical facility (e.g., an intensive care unit), may be in an earlystage of a vascular inflammatory disease, may be in a late stage of avascular inflammatory disease, or may have or be suspected of having abacterial infection. The subject may be a male, a female, a child, or aninfant.

The subject may begin to receive treatment within at least 48 hours, 36hours, 24 hours, 20 hours, 16 hours, 12 hours, 10 hours, or 6 hours ofpresentation to a health care professional or a medical facility. Insome examples, the subject may already be admitted to a health carefacility and is diagnosed as having a bacterial infection or issuspected of having a bacterial infection. The subject may receivetreatment prior to the presentation of any symptoms, prior to adiagnosis of having a vascular inflammatory disease, upon indicationthat subject has an increased risk of developing a vascular inflammatorydisease, at an intermediate stage of the disease (e.g., uponpresentation of one or more symptoms of a vascular inflammatorydisease), at a late stage of the disease (e.g., upon presentation of oneor more severe symptoms of a vascular inflammatory disease that mayrequire admission into a medical care facility), following diagnosis ofa bacterial infection, or upon presenting with one or more symptomswhich suggest the subject may have a bacterial infection. The treatmentmay be performed by a health care professional (e.g., a physician, anurse, and a physician's assistant) or by the subject.

A health care professional may assess the effect of the treatment byobserving or measuring one or more (e.g., two, three, four, or five)symptoms of a vascular inflammatory disease in a subject. Non-limitingsymptoms of vascular inflammatory diseases are described herein and maybe measured by physical examination. Additional molecular methods fordetermining the severity of a vascular inflammatory disease are known inthe art and may also be used to assess the efficacy of treatment in asubject (e.g., serum levels of C-reactive protein and inflammatorycytokines, such as TNF-α or IL-1). The health care professional mayadjust the frequency, dosage, or duration of treatment based on theassessment and measuring of one or more (e.g., two, three, four, orfive) symptoms of a vascular inflammatory disease in a subject duringtreatment.

A nucleic acid (e.g., a nucleic acid containing all or a part of thesequence of SEQ ID NO: 1) can be administered for prophylactic and/ortherapeutic treatments. In some embodiments, for therapeuticapplications, compositions are administered to a subject having avascular inflammatory disorder, or a subject who is at risk ofdeveloping a vascular inflammatory disorder, in an amount sufficient toreduce (the number, severity, and/or duration), or partially arrest, oneor more symptoms (e.g., two, three, four, five, or six) of a vascularinflammatory disorder; this can be called a therapeutically effectiveamount. For example, in some embodiments, pharmaceutical compositions(e.g., compositions containing a nucleic acid containing all or a partof SEQ ID NO: 1) are administered in an amount sufficient to decrease(e.g., a significant decrease, such as by at least 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or95%) leukocyte adhesion to the subject's endothelium, decrease (e.g., asignificant decrease, such as by at least 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%)leukocyte extravasion of the subject's endothelium, decrease (e.g., asignificant decrease, such as by at least 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%) theexpression (protein or mRNA) of VCAM-1, E-selectin, and/or ICAM-1 in thesubject's endothelium, (e.g., a significant decrease, such as by atleast 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, or 95%) reduce expression (protein or mRNA) ofimportin-α3 in the subject's endothelium, and/or decrease (e.g., asignificant decrease, such as by at least 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%)import of the p65 and/or p50 subunit of NF-κB into the nucleus of aendothelial cell in the subject compared to a control subject (e.g., asubject not administered the nucleic acid or the same subject prior totreatment with the nucleic acid).

The amount of the nucleic acid adequate to accomplish this is atherapeutically effective dose. The dosage schedule and amountseffective for this use, i.e., the dosing regimen, will depend upon avariety of factors, including the stage of the disease or condition, theseverity of the disease or condition, the general state of the patient'shealth, the patient's physical status, age, and the like. In calculatingthe dosage regimen for a patient, the mode of administration also istaken into consideration.

The dosage regimen also takes into consideration pharmacokineticsparameters well known in the art, i.e., the active agent's rate ofabsorption, bioavailability, metabolism, clearance, and the like (see,e.g., Hidalgo-Aragones, J. Steroid Biochem. Mol. Biol. 58:611-617, 1996;Groning, Pharmazie 51:337-341, 1996; Fotherby, Contraception 54:59-69,1996; Johnson, J. Pharm. Sci. 84:1144-1146, 1995; Rohatagi, Pharmazie50:610-613, 1995; Brophy, Eur. J. Clin. Pharmacol. 24:103-108, 1983;Remington: The Science and Practice of Pharmacy, 21st ed., 2005). Thestate of the art allows the heath care professional to determine thedosage regimen for each individual patient, active agent, and disease orcondition treated. Guidelines provided for similar compositions used aspharmaceuticals can be used as guidance to determine the dosageregiment, i.e., dose schedule and dosage levels, administered practicingthe methods described herein are correct and appropriate.

Single or multiple administrations of formulations can be givendepending on for example: the dosage and frequency as required andtolerated by the patient, the degree and amount of therapeutic effectgenerated after each administration (e.g., effect on one or moresymptoms of a vascular inflammatory disease), and the like. Theformulations should provide a sufficient quantity of active agent toeffectively treat, ameliorate, or delay the onset of a vascularinflammatory disease or one or more (e.g., two, three, four, five, orsix) of its symptoms.

One or more (e.g., two, three, four, or five) nucleic acids describedherein and, optionally, one or more (e.g., two, three, four, or five)additional anti-inflammatory agents (described below) can beadministered parenterally, intramuscularly, subcutaneously, arterially,intravenously, topically, orally, or by local administration, such as byaerosol or transdermally, to the subject.

The subject may be administered the nucleic acid once a day, twice aday, three times a day, four times a day, once a week, twice a week,three times a week, four times a week, once a month, twice a month,three times a month, four times a month, five times a month, six times amonth, seven times a month, eight times a month, bimonthly, once a year,twice a year, three times a year, or four times a year. A subject may beadministered the nucleic acid continuously (e.g., for at least 5minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 8hours, 12 hours, 24 hours, 36 hours, 48 hours, or 1 week) viaintravenous administration.

In alternative embodiments, pharmaceutical formulations for oraladministration are in a daily amount of between about 1 to 100 or moremg per kilogram of body weight per day. Lower dosages can be used, incontrast to administration orally, into the blood stream, into a bodycavity or into a lumen of an organ (e.g., into the lung). For example, adose of about 0.1 to 80, 0.1 to 70, 0.1 to 60, 0.1 to 50, 1 to 40, 1 to30, 1 to 20, 0.1 to 15, 0.1 to 10, or 0.1 to 5 mg per kg of body weightper day may be administered to the subject. Substantially higher dosagescan be used in topical or oral administration or administering bypowders, spray, or inhalation. Actual methods for preparing parenterallyor non-parenterally administrable formulations will be known or apparentto those skilled in the art and are described in more detail in suchpublications as Remington: The Science and Practice of Pharmacy, 21sted., 2005.

Various studies, including the animal model data provided herein, havereported successful mammalian dosing using nucleic acid sequences. Forexample, Esau C., et al., Cell Metabolism, 3(2):87-98, 2006, reporteddosing of normal mice with intraperitoneal doses of an miR-122 antisenseoligonucleotide ranging from 12.5 to 75 mg/kg twice weekly for 4 weeks.The mice appeared healthy and normal at the end of treatment, with noloss of body weight or reduced food intake. Plasma transaminase levelswere in the normal range (AST ¾ 45, ALT ¾ 35) for all doses with theexception of the 75 mg/kg dose of miR-122 ASO, which showed a very mildincrease in ALT and AST levels. They concluded that 50 mg/kg was aneffective, non-toxic dose. Another study by Krützfeldt J., et al.,Nature 438, 685-689, 2005, injected a nucleic acid to silence miR-122 inmice using a total dose of 80, 160 or 240 mg per kg body weight. Thehighest dose resulted in a complete loss of miR-122 signal. In yetanother study, locked nucleic acids (“LNAs”) were successfully appliedin primates to silence miR-122. Elmen et al., Nature 452, 896-899, 2008,report that efficient silencing of miR-122 was achieved in primates bythree doses of 10 mg per kg LNA-anti-miR, leading to a long-lasting andreversible decrease in total plasma cholesterol without any evidence forLNA-associated toxicities or histopathological changes in the studyanimals.

In some embodiments, the methods described herein can includeco-administration with other drugs or pharmaceuticals, e.g., one or more(e.g., two, three, four, or five) additional anti-inflammatory agents(e.g., corticosteroids, immunosuppressive agents, TNF-α antagonists,such as etanercept, infliximab, or adalimumab, and IL-1 antagonists,such as anakinra). For example, the provided nucleic acids can beco-administered with additional agents for treating or reducing risk ofdeveloping a vascular inflammatory disease described herein. Severalagents useful for the treatment of a vascular inflammatory disease areknown in the art.

The one or more additional anti-inflammatory agents may be administeredto the subject in a dose of between 0.1 to 100, 0.1 to 80, 0.1 to 70,0.1 to 60, 0.1 to 50, 1 to 40, 1 to 30, 1 to 20, 0.1 to 15, 0.1 to 10,or 0.1 to 5 mg per kg of body weight per day, depending on the specificagent and the route of administration. The one or more additionalanti-inflammatory agents may be formulated together with the one or morenucleic acids described herein in a single dosage form (e.g., an aerosolfor inhalation, a solid form for oral administration, or as a solutionfor intravenous administration). In some embodiments, the one or morenucleic acids described herein (e.g., a nucleic acid containing all or apart of SEQ ID NO: 1) may be administered in a separate dosage form fromthe one or more additional anti-inflammatory agents. In someembodiments, the subject is first administered at least one dose of anadditional anti-inflammatory agent prior to the administration of atleast one dose of the nucleic acids described herein. In someembodiments, the subject is first administered at least one dose of thenucleic acids described herein prior to the administration of at leastone dose of an additional anti-inflammatory agent. In some embodiments,the bioactive periods of the nucleic acid and the additionalanti-inflammatory agent overlap in the subject.

Methods of Decreasing NF-kB Signaling in a Cell

Also provided are methods for decreasing NF-κB signaling in a cell(e.g., an endothelial cell) by administering at least one nucleic acidthat contains all or a part of the sequence of mature miR-181b (SEQ IDNO: 1). The cell (e.g., an endothelial cell) may be present in a subjector may be a cell (e.g., an endothelial cell) that is present in vitro(tissue culture) or a cell that is removed from a subject and grown intissue culture (e.g., an ex vivo endothelial cell).

Cell in a Subject

In some embodiments, the cell (e.g., endothelial cell) is in a subject.For example, a health care professional may determine that a subject hasor may have an increased level of NF-κB signaling that contributes to apathophysiological condition in the subject (e.g., one or more of thediseases, e.g., a vascular inflammatory disease, described herein). Insuch instances, a health care professional may prescribe treatment ofthe subject according to the provided methods. The subject may bepresenting with one or more symptoms of such a condition (e.g., avascular inflammatory disease), may have been previously diagnosed ashaving such a condition (e.g., a vascular inflammatory disease), may beasymptomatic but at increased risk of later developing such a condition(e.g., a vascular inflammatory disease), may be in an early stage of thedisease (e.g., a vascular inflammatory disease), may in a late stage ofthe disease (e.g., a vascular inflammatory disease), may be admitted ina medical facility (e.g., a hospital, an intensive care unit, or anassisted care facility), may be diagnosed as having a bacterialinfection, or may be suspected of having a bacterial infection. Thesubject may also present with one or more (e.g., two, three, four, orfive) of the following features: increased (e.g., a significantincrease, such an increase of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%)leukocyte adhesion to the subject's endothelium, increased (e.g., asignificant increase, such an increase of at least 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or100%) leukocyte extravasion of the subject's endothelium, increased(e.g., a significant increase, such an increase of at least 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 100%) expression (protein or mRNA) of one or more ofVCAM-1, E-selectin, and ICAM-1 in the endothelium, increased (e.g., asignificant increase, such an increase of at least 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or100%) expression (protein or mRNA) of one or more genes regulated byNF-κB activity, increased (e.g., a significant increase, such anincrease of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) levels ofpro-inflammatory cytokines (e.g., IL-1 and TNF-α), and increased (e.g.,a significant increase, such an increase of at least 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or100%) levels of C-reactive protein. The subject may be a male, a female,a child, or an infant.

The subject may begin to receive treatment within at least 48 hours, 36hours, 24 hours, 20 hours, 16 hours, 12 hours, 10 hours, or 6 hours ofpresentation to a health care professional or a medical facility. Thesubject may receive treatment prior to the presentation of any symptoms,prior to a diagnosis of having a disease (e.g., a vascular inflammatorydisease), upon indication that subject has an increased risk ofdeveloping a disease (e.g., a vascular inflammatory disease), at anintermediate stage of the disease (e.g., following diagnosis or uponpresentation of one or more symptoms of a vascular inflammatorydisease), at a late stage of the disease (e.g., upon presentation of oneor more severe symptoms of a vascular inflammatory disease that mayrequire admission into a medical care facility), following diagnosis ofa bacterial infection, or upon suspicion of having a bacterialinfection. The treatment may be performed by a health care professional(e.g., a physician, a nurse, and a physician's assistant) or by thesubject.

A health care professional may assess the effect of the treatment byobserving or measuring one or more (e.g., two, three, four, or five)symptoms of a disease (e.g., a vascular inflammatory disease) in asubject. Non-limiting symptoms of vascular inflammatory diseases aredescribed herein and may be measured by physical examination. Additionalmolecular methods for determining the severity of a disease (e.g., avascular inflammatory disease) are known in the art and may also be usedto assess the efficacy of treatment in a subject (e.g., serum levels ofC-reactive protein and pro-inflammatory cytokines, such as IL-1 andTNF-α). In addition, the efficacy of treatment may be assessed bydetermining whether there is: a decrease (e.g., a significant decrease,such a decrease of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%) in leukocyte adhesion inthe subject's endothelium, a decrease (e.g., a significant decrease,such a decrease of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%) in leukocyte extravasionof the subject's endothelium, a decrease (e.g., a significant decrease,such a decrease of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%) in NF-κB-induced geneexpression, a decrease (e.g., a significant decrease, such a decrease ofat least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%,75%, 80%, 85%, 90%, or 95%) in the expression levels (protein or mRNA)of importin-α3, a decrease (e.g., a significant decrease, such adecrease of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%) in the expression levels(protein or mRNA) of one or more of ICAM-1, VCAM-1, and E-selectin, or adecrease (a decrease (e.g., a significant decrease, such a decrease ofat least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%,75%, 80%, 85%, 90%, or 95%) in the expression (protein or mRNA) of oneor more of the genes listed in FIG. 9A, as compared to control subjectnot receiving the nucleic acid (e.g., a subject not diagnosed or notpresenting with one or more symptoms of a vascular inflammatory diseaseor the same subject prior to administration of the nucleic acid). Thehealth care professional may adjust the frequency, dosage, or durationof treatment based on the assessment and measuring of one or more (e.g.,two, three, four, or five) symptoms of a disease (e.g., a vascularinflammatory disease) in a subject during treatment.

Any of the nucleic acids described herein may be administered in thesemethods. In addition, any of the dosing and administration schedulesdescribed herein may be used in these methods. As described herein, inaddition to administering one or more of the nucleic acids describedherein, one or more anti-inflammatory agents may also be administered tothe subject. Any of the formulations, compositions, administrationschedules, and dosing described herein may be used in these methodswithout limitation.

Cell In Vitro and Ex Vivo

As indicated above, the cell (e.g., an endothelial cell) administered anucleic acid containing all or a part of the sequence of mature miR-181b(SEQ ID NO: 1) may be present in vivo or may have previously beenremoved from a subject and treated with the nucleic acid in tissueculture (ex vivo). A cell (e.g., an endothelial cell) removed from thesubject and administered the nucleic acid in tissue culture may beimplanted into a subject following said administration. For example, theremoved cell (e.g., endothelial cell) may be cultured in the presence ofthe nucleic acid for at least 6 hours, 12 hours, 18 hours, 24 hours, 48hours, 3 days, 4 days, 5 days, 6 days, or 1 week before implanting thecell back into the subject. The cell removed from the subject may alsobe transfected with another nucleic acid to induce expression of atherapeutic protein (e.g., a protein that decreases inflammation in asubject).

One skilled in the art would readily be able to determine the amount ofthe nucleic acid required to affect a decrease in NF-κB signaling in thecell (e.g., endothelial cell). Exemplary methods are described in thespecification and are known in the art. For example, a decrease in NF-κBsignaling may be observed by a: decrease (e.g., a significant decrease,such a decrease of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%) in NF-κB-induced geneexpression, a decrease (e.g., a significant decrease, such a decrease ofat least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%,75%, 80%, 85%, 90%, or 95%) in nuclear translocation of NF-κB, and adecrease (e.g., a significant decrease, such a decrease of at least 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%,85%, 90%, or 95%) in NF-κB binding to its promoter elements compared tocontrol cells (e.g., control cells treated with an agent that stimulatesNF-κB signaling activity, such as TNF-α).

Any of the nucleic acids or compositions described herein may be used inthese methods.

EXAMPLES

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

Example 1 MiR-181b is a Critical Regulator of NF-κB-Mediated EndothelialCell Activation and Vascular Inflammation In Vivo

MiR-181b Expression in Endothelial Cells is Regulated by TNF-α.

In an attempt to identify how pro-inflammatory stimuli regulateendothelial function, microarray miR profiling studies were undertakenusing RNA from HUVECs exposed to vehicle alone or TNF-α for 24 h.Increased expression of miR-181b was observed in association with TNF-αtreatment in HUVECs. Using real-time PCR analysis, the induction ofmiR-181b in HUVECs in response to TNF-α exposure was verified: anincrease in expression by about 1.3-fold at 24 h (FIG. 1A).Surprisingly, earlier time points (at 1 and 4 h) revealed that TNF-αinhibited miR-181b expression by 31% and 24%, respectively (FIG. 1A).

MiR-181b belongs to the miR-181 family, which is comprised of fourmature miRNAs: miR-181a, miR-181b, miR-181c, and miR-181d. These maturesequences are encoded by six primary miRNA sequences located on threedifferent chromosomes. The expression of miR-181b was determined to beabout 12-fold higher than that of miR-181a, and 274-fold higher thanthat of miR-181c (FIG. 1B). Since the level of primary (pri)-miR-181dwas very low, the level of this mature miR-181d was not examined.Collectively, these data suggest that miR-181b is the dominantlyexpressed miR-181 family member in HUVECs and that it has a bi-modalexpression pattern in response to stimulation by the inflammatorycytokine TNF-α.

MiR-181b Inhibits TNF-α-Induced Expression of Adhesion Molecules andInhibits Leukocyte Adhesion to Activated EC Monolayers

To assess the potential role of miR-181b in endothelial activation, theeffect of miR-181b on TNF-α-induced gene expression was examined inHUVECs using gain- and loss-of-function experiments. Over-expression ofmiR-181b inhibited TNF-α-induced VCAM-1, E-selectin, and ICAM-1 proteinexpression by 89%, 52%, and 36%, respectively, while, miR-181binhibitors (complementary antagonist) increased their expression by138%, 53%, and 31%, respectively (FIG. 1C). Consistent with theseresults, the mRNA levels of VCAM-1, E-selectin, and ICAM-1 were lower incells over-expressing miR-181b than in cells over-expressing the miRNAnegative control; moreover, these mRNA levels were higher in thepresence of the miR-181b inhibitor (FIG. 1D). After 1-h TNF-α treatment,cells over-expressing miR-181b exhibited reduced mRNA levels of VCAM-1,E-selectin, and ICAM-1 (by 69%, 65%, and 57%, respectively); after 4 hof TNF-α treatment, the levels were 74%, 41%, and 17%, respectively. Incontrast, in cells transfected with miR-181b inhibitors, TNF-α-inducedVCAM-1 mRNA was increased by 62% after 1-h TNF-α treatment, and after 3h of TNF-α treatment, VCAM-1, E-selectin, and ICAM-1 mRNA levels wereincreased by 35%, 52%, and 34%, respectively (FIG. 1D). The effects ofmiR-181b on levels of soluble VCAM-1, E-selectin, and ICAM-1 in theculture medium, as measured by ELISA, were also consistent with itseffects on the mRNA and protein expression of these adhesion molecules(FIG. 1E). Likewise, miR-181b also reduced VCAM-1 expression at bothprotein and mRNA level in HUVECs in response to LPS treatment (FIG.2A-C) and the expression of E-selectin mRNA and ICAM-1 mRNA (FIG. 2C).

Considering the finding that VCAM-1 expression was most sensitive tomiR-181b, experiments were performed to determine whether the VCAM-1gene might be a direct target of the miR. However, over-expression ofmiR-181b did not reduce the luciferase activity of a VCAM-1 3′-UTRconstruct, suggesting that the VCAM-1 3′-UTR is not directly targeted bymiR-181b (FIG. 2D). Since VCAM-1, E-selectin, and ICAM-1 are typicalpro-inflammatory molecules induced by TNF-α, these data suggested thatmiR-181b may be involved in the regulation of endothelial cellactivation. To investigate whether miR-181a, the next highest expressedmiR-181 family member in HUVECs, was able to elicit the same effect onTNF-α-induced gene expression as miR-181b did, HUVECs were transfectedwith miRNA negative control, miR-181a, or miR-181b at differentconcentrations in the presence or absence of TNF-α, and harvested forWestern blot analysis of VCAM-1 expression. As shown in FIG. 2E,miR-181a inhibited TNF-α-induced VCAM-1 expression to a lesser extentthan miR-181b at all concentrations examined.

In response to endothelial cell activation, adhesion molecules, such asVCAM-1, E-selectin, and ICAM-1, act to initiate, promote, and sustainleukocyte attachment to the vascular endothelium. To determine thefunctional consequence of miR-181b effects on adhesion moleculeexpression, in vitro cell adhesion assays were performed to assessleukocyte-endothelial cell interactions. As expected, TNF-α treatmentmarkedly increased the adhesion capabilities of THP-1 cells to HUVECstransfected with non-specific (NS) control miRNA mimics. However,adhesion was markedly reduced (by 44%) with miR-181b over-expression,whereas inhibition of miR-181b increased the adherence by 50% (FIG. 1F).Taken together, these findings show that miR-181b is able to negativelyaffect the expression of key adhesion molecules induced bypro-inflammatory stimuli and that miR-181b dynamically regulatesleukocyte adhesion to stimulated EC monolayers.

MiR-181b Suppresses TNF-α-Induced Expression of Adhesion Molecules InVivo.

Experiments were performed to determine whether systemic administrationof miR-181b could inhibit TNF-α-induced gene expression in vivo.Atelocollagen, a highly purified bovine type I collagen withoutimmunogenicity, has been successfully used to deliver plasmid DNA,siRNA, and miRNAs into cells in vitro and in vivo (Ochiya et al., NatureMed. 5:707-710, 1999; Sano et al., Adv. Drug Deliv. Rev. 55:1651-1677,2003; Takeshita et al., Methods Mol. Biol. 487:83-92, 2009; Takeshita etal., Proc. Natl. Acad. Sci. U.S.A. 102:12177-12182, 2005; Takeshita etal., Mol. Ther. 18:181-187, 2010; Tazawa et al., Proc. Natl. Acad. Sci.U.S.A. 104:15472-15477, 2007). To assess the effects of miR-181b mimicson TNF-α-induced expression of VCAM-1 in vivo, miR-181b or anon-specific control mimic, was admixed with atelocollagen and tailvein-injected 24 h prior to TNF-α treatment. VCAM-1 protein expressionwas first examined in lung tissues. At 4 h after TNF-α i.p. injection,VCAM-1 was found to be induced by ˜3.4-fold in lung tissues in thepresence of non-specific control mimics. In contrast, administration ofmiR-181b potently reduced the induction of VCAM-1 protein expression (to˜2-fold) (FIG. 3A) and VCAM-1 mRNA expression in lung, aorta, heart,liver, and spleen (FIG. 3B). The expression of E-selectin mRNA was alsosignificantly reduced in lung, liver, and spleen (FIG. 4A), whereasICAM-1 mRNA was reduced only in spleen (FIG. 4B).

To further verify the observed effects on VCAM-1 expression, sections oflung and descending aorta were examined by immunohistochemicaltechniques. The endothelium of lung and aorta from mice injected withmiRNA negative control displayed robust VCAM-1 expression in response toTNF-α (FIG. 3C-E). In contrast, systemic administration of miR-181bmimics reduced the induction of VCAM-1 expression in the endothelium inlung and aorta (by ˜86% and ˜80%, respectively) (FIG. 3C-E). Notably,the expression of miR-181b in the intima of aortae excised from miceinjected with miR-181b was ˜8-fold higher than in mice injected withmiRNA negative control, as measured by real-time qPCR (FIG. 4C). Therewere no significant differences of miR-181b expression levels in themedia and adventitia excised from mice injected with miR-181b or miRNAnegative control (FIG. 4C). In summary, these data demonstrated thatsystemically administered miR-181b mimics were efficiently enriched inendothelial cells, and inhibited expression of TNF-α-induced adhesionmolecules in vivo.

MiR-181b Inhibits the NF-κB Signaling Pathway in Activated EndothelialCells.

In response to pro-inflammatory stimuli, both the NF-κB andmitogen-activated protein (MAP) kinase pathway are involved in theinflammatory responses in endothelial cells (Hoefen et al., Vascul.Pharmacol. 38:271-273, 2002; Kempe et al., Nucleic Acids Res.33:5308-5319, 2005). To examine whether miR-181b affects NF-κBactivation, experiments were performed to test whether miR-181b has anyeffect on the NF-κB concatemer and VCAM-1 promoter-reporter. As shown inFIG. 3A, treatment of HUVECs with TNF-α induced the activity of both theNF-κB concatemer and the VCAM-1 promoter-reporter, and co-transfectionof miR-181b significantly attenuated this induction. In contrast,inhibition of miR-181b potentiated TNF-α-induced activity (FIG. 5A). Theeffect of miR-181b on NF-κB nuclear accumulation was then determined byimmunostaining for p65. A nearly 40% reduction in p65 nuclear stainingwas observed in HUVECs transfected with miR-181b, as compared to cellstransfected with a miRNA negative control (FIG. 5B). After its releasefrom the IκB complex, translocation of NF-κB from the cytoplasm to thenucleus is an essential step for the activation of NF-κB target genes(Oeckinghaus et al., Cold Spring Harbor Perspect. Biol. 1:a000034, 2009;Vallabhapurapu et al., Annu. Rev. Immunol. 27:693-733, 2009), an effectthat can be revealed by detection of the p50 and p65 protein levels incytoplasmic and nuclear fractions. As shown in FIG. 5C, HUVECsover-expressing miR-181b exhibited reduced p65 and p50 expression in thenuclear fraction, whereas the cytoplasmic fraction had increased p65 andp50 expression. Importantly, no significant differences between miR-181band the miRNA negative control on the expression of upstream componentsof the NF-κB pathway was observed, including phosphorylated IκBα: aneffect suggesting it is unlikely that miR-181b affects cell surfacereceptors or activation of the IKK complex. Since several MAP kinaseshave been implicated in TNF-α-induced expression of adhesion molecules,experiments were performed to test whether miR-181b had any effect onthe activation of three MAP kinases (extracellular signal-regulatedkinase (ERK), p38, and Jun-amino-terminal kinase (JNK)) in response toTNF-α. As shown in FIG. 6, the phosphorylation of ERK, p38, and JNK wasrobustly induced and peaked at 15 min after TNF-α treatment. However,miR-181b over-expression had no effect on their phosphorylation at 5,15, and 30 min after TNF-α treatment. These data suggest that theinhibitory role of miR-181b on TNF-α-induced gene expression isprimarily due to its effects on the NF-κB signaling pathway byrepressing NF-κB nuclear translocation.

MiR-181b Directly Targets Expression of Importin-α3, a Protein Criticalfor NF-κB Nuclear Translocation.

Previous studies have shown that NF-κBs are transported into the nucleusvia a subset of importin-α molecules (Fagerlund et al., J. Biol. Chem.280:15942-15951, 2005; Fagerlund et al., Cell Signal. 20:1442-1451,2008). There are six importin-α paralogs in humans (importin-α1, -α3,-α4, -α5, -α6, and -α7) that are characterized by distinct affinities totheir substrates (Kohler et al., Mol. Cell. Biol. 19:7782-7791, 1999).In miR-181b over-expressing cells, importin-α3 expression, but not thatof importin-α1 or importin-α5, was reduced by 46% in the presence ofTNF-α (FIG. 7A). Over-expression of miR-181b inhibited the activity of aluciferase reporter construct containing importin-α3 3′-UTR in a dosedependent manner (FIG. 7B). In contrast, the activity of luciferaseconstructs containing the 3′-UTR of importin-α1, -α4, or -α5 was notinhibited by over-expressed miR-181b (FIG. 7C).

The rna22 miRNA target detection and predication algorithm allows forseed mismatches between mature miRNA and targeting mRNA sequence(Miranda et al., 2006), and has been successfully used to identifydirect targets that were not predicted by other algorithms (Lal et al.,Mol. Cell 35:610-625, 2009). To identify additional miR-181b bindingsites that may exist in the 3′-UTR of importin-α3, the rna22 predictionalgorithm was applied. The results identified eight potential miR-181bbinding sites in the region of interest (FIG. 8A). Over-expression ofmiR-181b decreased luciferase activity by 31% and 20%, respectively, forluciferase-reporter constructs containing binding site 1 and site 2, butnot for any of the other potential binding sites (FIG. 7D).Site-directed mutations of binding site 1 and site 2 rescued themiR-181b-mediated inhibitory effects on both of these constructs (FIG.7D). Interestingly, the mRNA level of importin-α3 was not altered bymiR-181b over-expression (FIG. 8B): an effect indicating that thereduction of importin-α3 at protein level is likely due to translationinhibition and not mRNA decay.

To further verify that miR-181b directly targets importin-α3, Argonaute2(AGO2) microribonucleoprotein immunoprecipitation (miRNP-IP) studieswere performed to assess whether importin-α3 mRNA is enriched in theRNA-induced silencing complex following miR-181b over-expression. Anapproximately 4-fold enrichment of importin-α3 mRNA was observed afterAGO2 IP in the presence of miR-181b, as compared to that with the miRNAnegative control (FIG. 7E). In contrast, AGO2 IP did not enrich the mRNAfor Smad1, a gene that was not predicted to be a miR-181b target (FIG.7E). Moreover, expression of importin-α3 lacking its 3′-UTR was able torescue the inhibitory effect of miR-181b on NF-κB activation (FIG. 7F).Collectively, these data suggest that miR-181b inhibits the NF-κBsignaling pathway by directly targeting importin-α3 expression.

MiR-181b Over-Expression Inhibited an Enriched Set of NF-κB RegulatedGenes in Endothelial Cells.

To systemically identify targets and biological processes regulated bymiR-181b, the gene expression profiles of HUVECs transfected with miRNAnegative control or miR-181b were comparatively analyzed using Agilentwhole human genome microarrays. Transfected HUVECs were treated withTNF-α for 4 h and total RNA was isolated and processed for gene chipanalysis. Out of the ˜44,000 transcripts screened, 841 genes weredown-regulated and 928 genes were up-regulated by at least 1.5-fold inmiR-181b over-expression cells, as compared with control cells. Over 200of those genes are known to be NF-κB-regulated. Moreover, 29 of thesegenes are associated with inflammation, and were all inhibited byover-expression of miR-181b. These reduced gene expression changes wereverified by qPCR analysis (FIGS. 9A and 9B). PAI-1, COX-2, CX3CL-1, andVCAM-1 were chosen for further analysis by Western blot to verifyconcordant directional change in protein levels (FIG. 9C).

Among the 29 NF-κB regulated genes inhibited by miR-181b, some representpotential direct targets of miR-181b; for example, TIMP3 has beenpredicted as a direct target by TargetScan5.1 (Lewis et al., Cell120:15-20, 2005) and by PicTar (Krek et al., Nature Genet. 37:495-500,2005) algorithms, and EGR3 has been predicted by PicTar (Krek et al.,2005). Thus, the possibility that the observed reduction of EGR3 andTIMP3 may be a consequence of miR-181b's inhibitory effect on NF-κBactivation and/or a direct effect of miR-181b on the 3′UTRs of thesegenes cannot be ruled out. To identify highly-regulated biologicalprocesses in miR-181b over-expressing cells, gene set enrichmentanalysis (GSEA), a computational method that determines if a defined setof genes shows significant differences between two biological states(Subramanian et al., Proc. Natl. Acad. Sci. U.S.A. 102:15545-15550,2005), was performed. Six enriched biological processes weresignificantly represented by the reduced genes in miR-181bover-expressing cells: response to cytokine stimulus; positiveregulation of cell migration; regulation of inflammatory response;inflammatory response; chemotaxis; and IκB kinase/NF-κB cascade (FIG.9D). An abundance of targets that interconnected with the NF-κBsignaling pathway were also observed using the Ingenuity web-basedpathway analysis program. Taken together, these data suggest thatmiR-181b selectively suppressed an enriched set of NF-κB regulated genesand components of inflammatory signaling pathways in response to TNF-αin endothelial cells.

MiR-181b Inhibits LPS-Induced Endothelial Cell Activation, LeukocyteAccumulation, and Lung Airway Inflammation.

Sepsis is a severe medical condition with increasing incidence over thepast few decades. Patients with sepsis are also at risk for otherlife-threatening complications, such as multisystem organ failure. Theendotoxin LPS is a component of the outer membrane of Gram-negativebacteria, which plays a significant role in the pathogenesis of about25%-30% sepsis (Annane et al., Lancet 365:63-78, 2005). LPS induces therelease of critical pro-inflammatory cytokines including TNF-α, andelicits a systemic inflammatory response syndrome. During sepsis,activation of the vascular endothelium plays a critical role in therecruitment of neutrophils and monocytes/macrophages, and subsequentexacerbation of the inflammatory response (Aird, Blood 101:3765-3777,2003; Woodman et al., Am. J. Physiol. Heart Circ. Physiol.279:H1338-1345, 2000). To test if miR-181b contributes to this process,experiments were performed to determine whether in vivo over-expressionof miR181b could reduce leukocyte recruitment and endothelial cellactivation in a systemic LPS-induced mouse model of vascularinflammation. In vivo intravenous administration of miR-181b mimicsreduced the induction of VCAM-1 expression in response to LPS by 60%(FIG. 10A, 10E). Analysis of lung sections taken from those miceinjected with miR-181b revealed a significant reduction of CD45 (commonleukocyte antigen)- and Gr-1 (predominantly neutrophil)-positiveleukocytes (by 54% and 58%, respectively) in response to LPS treatment(FIGS. 10A, 10C, and 10D). Importantly, the number of Gr-1-positiveleukocytes adherent to the pulmonary vascular endothelium was reduced47% by miR-181b overexpression (FIGS. 11C and 11D). Reduced interstitialedema in the lungs of mice in which miR-181b was over-expressed was alsoobserved (FIG. 10A). Lung injury scores were calculated, and the lungsfrom mice injected with miR-181b showed less damage (FIG. 10B).

Myleoperoxidase activity, reflecting the presence of peroxidase enzymeexpressed most abundantly in neutrophils, was also reduced by miR-181bover-expression (FIG. 10F). Taken together, these results demonstrate acritical role for miR-181b in endotoxin-induced endothelial activationand leukocyte accumulation, and suggest that over-expression of miR-181bcould represent a new class of therapeutic avenues for limitingendotoxin-induced vascular inflammation.

To test if miR-181b contributes to this process, experiments wereperformed to determine whether the level of endothelium miR-181b couldbe affected in response to LPS, and whether in vivo over-expression ofmiR181b could reduce leukocyte recruitment and EC activation in asystemic LPS-induced mouse model of vascular inflammation. Four hoursafter LPS, the level of miR-181b was reduced 50% in freshly isolatedaortic intima, while TNF-α treatment caused 47% reduction (FIG. 11A).The mRNA of VCAM-1 was induced about 5.0-fold and 7.2-fold by TNF-α andLPS in freshly isolated aortic intima respectively (FIG. 11B).

Sustained endothelial cell activation adversely contributes to thepathogenesis of both acute and chronic inflammatory disease states. Asdemonstrated herein, miR-181b is dynamically regulated in response topro-inflammatory stimuli and functions to suppress the expression of anenriched set of NF-κB target genes associated with inflammatory diseasestates, such as adhesion molecules (e.g., VCAM-1 and E-selectin),chemokines, and chemokine receptors (e.g., CCL1, CCL7, CX3CL1, CXCL1,and CCR2), and other key inflammatory mediators (e.g., COX-2, PAI-1,EGR, and TRAF1). Furthermore, the data show that miR-181b directlytargets importin-α3, an effect that inhibits nuclear accumulation of p50and p65. Finally, administration of miR-181b mimics also reducedexpression of adhesion molecules, leukocyte accumulation, and vascularinflammation in vivo. As such, these studies identify miR-181b as aregulator of endothelial cell activation in vitro and in vivo.

miR-181b is identified herein as a cytokine-responsive miRNA thatregulates the expression of key NF-κB-regulated genes involved in theendothelial response to inflammation in vitro and in vivo. Thesefindings also revealed a novel and unexpected mechanistic role for thismiRNA in targeting downstream NF-κB signaling by directly targetingimportin-α3. These studies support the use of miR-181b as an agent thatcan control critical aspects of endothelial cell homeostasis underphysiologic or pathologic conditions.

Materials and Methods

Reagents and Antibodies

Cy3 dye-labeled Pre-miR negative control #1 (AM17120), Pre-miR miRNAprecursor molecules-negative control #1 (AM17110), Pre-miR miRNAprecursor molecules-miR-181b (PM12442), Anti-miR miRNAinhibitors-negative control #1 (AM17010), and miR-181b inhibitor(AM12442) were from Ambion. For in vivo studies, oligomers with the samesequence were synthesized on a larger scale. Anti-p65 (sc-8008),anti-p50 (sc-8414), anti-IKKα (sc-7182), anti-IKKγ (sc-8330), goatanti-mouse VCAM-1 (sc-1504), mouse anti-human VCAM-1 (sc-13160), goatanti-mouse IgG-HRP (sc-2005), goat anti-rabbit IgG-HRP (sc-2004), goatanti-chicken IgY-HRP (sc-2901), donkey anti-goat IgG-HRP (sc-2020) werefrom Santa Cruz Biotechnology. Cy5-conjugated goat anti-mouse IgG wasfrom Jackson ImmunoResearch. Anti-phospho-p38 MAPK (Thr180/Tyr182)(4511), anti-phospho-SAPK/JNK (Thr183/Tyr185) (4668),anti-phospho-p44/42 MAPK (Thr202/Tyr204) (4370), anti-p38 MAPK (9212),anti-SAPK/JNK (9258), anti-IκBα (4814), anti-β-actin (4970),anti-Histone H3 (9715), anti-IKKβ (2370), anti-p44/42 (9107),anti-phospho-IκBα (2859), anti-Cox-2 (4842) were from Cell Signaling.Anti-importin-α1 (D168-3), anti-importin-α3 (D169-3), anti-importin-α5/7(D170-3) were from MBL, Medical & Biological Laboratories. Monoclonalanti-human E-selectin (CD62E) (S9555) was from Sigma. Anti-humanE-selectin monoclonal antibody (BBA16), anti-rat IgG-HRP (HAF005),anti-human ICAM-1/CD54 Clone BBIG-I1 (BBA3), anti-human PAI-1 (MAB1786)were from R&D Systems. Anti-CX3CL-1 (14-7986) was from eBioscience.Anti-Ly-6G and Ly-6C/Gr-1 (550291), and anti-CD45 (550539) were from BDPharmingen.

Cell Culture and Transfection

THP-1 cells were from ATCC, and cultured in ATCC-formulated RPMI-1640medium (30-2001) supplemented with 10% fetal bovine serum and 0.05 mM2-mercaptoethanol. HUVECs were obtained from Lonza (cc-2159) andcultured in endothelial cell growth medium EGM-2 (cc-3162). Cellspassaged less than five times were used for all experiments.Lipofectamine 2000 transfection reagent from Invitrogen was used fortransfection, following the manufacturer's instructions. Cells wereallowed to grow for 36 h before treatment with 10 ng/ml recombinanthuman TNF-α from R & D Systems (210-TA/CF) for various times, accordingto the experiment: Western blot, 8 h; real-time qPCR, 1, 3, 4, or 16 h;or ELISA, 16 h.

Constructs

The human miR-181b gene, including 96 bp upstream and 241 bp downstreamflanking regions of its stem loop sequence, was amplified by PCR fromhuman genomic DNA (Promega) using Platinum PCR SuperMix High FidelityTaq-based enzyme mix (Invitrogen). The resultant fragment was subclonedinto the pcDNA3.1 (+) vector to generate the pcDNA3.1(+)-miR-181bplasmid. Primers 5′-CCCAAGCTTTGATTGTAC CCTATGGCT-3′ (forward; SEQ ID NO:4) and 5′-CGGGGTACCTGTACGTTTGATGG ACAA-3′ (reverse; SEQ ID NO: 5) wereused to amplify human miR-181b coding sequence.

The 3′-UTR of genes for importin-al, importin-α3, importin-α4,importin-α5, and VCAM-1 were amplified from human genomic DNA and clonedinto the pMIR-REPORT Luciferase vector, between SacI and MluIrestriction sites (importins) or MluI and HindIII restriction sites(VCAM-1). Putative miR-181b binding sites in the importin-α3 gene 3′-UTRwere predicted by the rna22 algorithm. Individual wild-type or mutantbinding site sequence was generated by annealing the forward and reverseoligonucleotides containing SpeI and HindIII sticky ends, followed by T4Polynucleotide Kinase (New England Biolabs) phosphorylation. Thedouble-stranded oligonucleotides were ligated into the pMIR-REPORTLuciferase vector, between SpeI and HindIII restriction sites, using T4DNA ligase (New England Biolabs). A construct containing the openreading frame cDNA of importin-α3 was purchased from OriGene. Theprimers used are listed in Table 1 below.

TABLE 1 Primers used for Studies Name Sequence (5′ −> 3′) For Real mouse VCAM-1 F: GTTCCAGCGAGGGTCTACC  Time qPCR (SEQ ID NO: 6)mouse VCAM-1 R: AACTCTTGGCAAACATTAGGTGT  (SEQ ID NO: 7) mouse E-ATGCCTCGCGCTTTCTCTC  selectin F: (SEQ ID NO: 8) mouse E-GTAGTCCCGCTGACAGTATGC  selectin R: (SEQ ID NO: 9) mouse ICAM-1 F:GTGATGCTCAGGTATCCATCCA  (SEQ ID NO: 10) mouse ICAM-1 R:CACAGTTCTCAAAGCACAGCG  (SEQ ID NO: 11) mouse actin F:GAAATCGTGCGTGACATCAAAG  (SEQ ID NO: 12) mouse actin R:TGTAGTTTCATGGATGCCACAG  (SEQ ID NO: 13) human PAI-1 F:CATCCCCCATCCTACGTGG  (SEQ ID NO: 14) human PAI-1 R:CCCCATAGGGTGAGAAAACCA  (SEQ ID NO: 15) human VCAM-1 F:GCTGCTCAGATTGGAGACTCA  (SEQ ID NO: 16) human VCAM-1 R:CGCTCAGAGGGCTGTCTATC  (SEQ ID NO: 17) human E- AATCCAGCCAATGGGTTCG selectin F: (SEQ ID NO: 18) human E- GCTCCCATTAGTTCAAATCCTTCT selectin R: (SEQ ID NO: 19) human ICAM-1 F: TCTGTGTCCCCCTCAAAAGTC (SEQ ID NO: 20) human ICAM-1 R: GGGGTCTCTATGCCCAACAA  (SEQ ID NO: 21)human GAPDH F: ATGGGGAAGGTGAAGGTCG  (SEQ ID NO: 22) human GAPDH R:GGGGTCATTGATGGCAACAATA  (SEQ ID NO: 23) mouse importin-CCAGTGATCGAAATCCACCAA  α3 F (SEQ ID NO: 24) mouse importin-CGTTTGTTCAGACGTTCCAGAT  α3 R: (SEQ ID NO: 25) human importin-TCCAGTGATCGAAATCCACCA  α3 F: (SEQ ID NO: 26) human importin-CATTGGACTGAACTACTGCTTGA  α3 R: (SEQ ID NO: 27) human Smad1 F:GATGCCAGGTAGGTTGGAATG  (SEQ ID NO: 28) human Smad1 R:CGTGACACTGTGATAACACTGT  (SEQ ID NO: 29) Oligo- Site1 forward:CTAGTCTTGCTATGAAGCAGTGTGTGAAA nucleotides (SEQ ID NO: 30) Site1 reverse:AGCTTTTCACACACTGCTTCATAGCAAGA (SEQ ID NO: 31) Site2 forward:CTAGTATGGACAATGTTGAATGAATGTCA (SEQ ID NO: 32) Site2 reverse:AGCTTGACATTCATTCAACATTGTCCATA (SEQ ID NO: 33) Site3 forward:CTAGTCTGTGTACGAGAGCGTGGTTGTGA (SEQ ID NO: 34) Site3 reverse:AGCTTCACAACCACGCTCTCGTACACAGA (SEQ ID NO: 35) Site4 forward:CTAGTTGGTTTACTCTGCAGCCTGTGTTA (SEQ ID NO: 36) Site4 reverse:AGCTTAACACAGGCTGCAGAGTAAACCAA (SEQ ID NO: 37) Site5 forward:CTAGTTGCATTTGCACCAGATGAATGTTA (SEQ ID NO: 38) Site5 reverse:AGCTTAACATTCATCTGGTGCAAATGCAA (SEQ ID NO: 39) Site6 forward:CTAGTTTTCCCTCAAAATAGACTGTGTTA (SEQ ID NO: 40) Site6 reverse:AGCTTAACACAGTCTATTTTGAGGGAAAA (SEQ ID NO: 41) Site7 forward:CTAGTATACCGTGCTGTGTTTAAATGTTA (SEQ ID NO: 42) Site7 reverse:AGCTTAACATTTAAACACAGCACGGTATA (SEQ ID NO: 43) Site8 forward:CTAGTCTTCCCCTTTGAGCACAAGTGTTA (SEQ ID NO: 44) Site8 reverse:AGCTTAACACTTGTGCTCAAAGGGGAAGA (SEQ ID NO: 45) Site1mut forward:CTAGTCTTGCTATGATAAAGCTTCTGAAA (SEQ ID NO: 46) Site1mut reverse:AGCTTTTCAGAAGCTTTATCATAGCAAGA (SEQ ID NO: 47) Site2mut forward:CTAGTAGGCTGAATCTTGCCAACATCACA (SEQ ID NO: 48) Site2mut reverse:AGCTTGTGATGTTGGCAAGATTCAGCCTA (SEQ ID NO: 49) For cloning Importin-ACGAGCTCATCATGTAGCTGAGACATAAA 3′-UTR α1 forward: TTTG (SEQ ID NO: 50)Importin- ATAACGCGTAGAAAAGGGTGGACTTGAATGT α1 reverse: (SEQ ID NO: 51)Importin- ACGAGCTCAAAGATGTTGTGGAAGTTAGG α3 forward: (SEQ ID NO: 52)Importin- ATAACGCGTCACAGCACGGTATTCTACCAC α3 reverse: (SEQ ID NO: 53)Importin- ACGAGCTCATTCAGTTGAGTGCAGCATC α4 forward: (SEQ ID NO: 54)Importin- ATAACGCGTCCTCTACACAGATCCCTGTC α4 reverse: (SEQ ID NO: 55)Importin- ACGAGCTCAGCAATACTCTGCTTTCACG α5 forward: (SEQ ID NO: 56)Importin- ATAACGCGTGATTAGAATCGAGCTGCACC α5 reverse: (SEQ ID NO: 57)VCAM-1 forward: TCGACGCGTGCAAATCCTTGATACTGC (SEQ ID NO: 58)VCAM-1 reverse: CCCAAGCTTATTGGGAAAGTTGCACAG (SEQ ID NO: 59)Luciferase Reporter Assays

HUVECs were plated (50,000/well) in triplicate on a 12-well plate. Aftergrowing to 70-80% confluency, cells were transfected with 200 ng of theindicated reporter constructs and 100 ng β-galactosidase (gal)expression plasmids. MiR-181b mimics or inhibitors were co-transfectedat 10 or 50 nM final concentration where indicated; after 36 hincubation, cells were treated with 10 ng/ml TNF-α for 8 h. In someexperiments, pcDNA3.1-miR-181b or empty vector were co-transfected with200 ng reporter constructs and 100 ng β-gal expression plasmids. Forrescue studies, NF-κB concatemer luciferase reporter was co-transfectedwith pcDNA3.1-miR-181b or empty vector in the presence or absence ofopen reading frame cDNA of importin-α3 into 293T cells. Transfectedcells were collected in 200 μL Reporter Lysis Buffer (Promega). Theactivity of luciferase and β-gal were measured. Each reading ofluciferase activity was normalized to the β-gal activity read for thesame lysate.

Enzyme-Linked Immunosorbent Assays (ELISAs)

HUVECs were transfected with control miRNAs, miR-181b at the finalconcentration of 10 nM, control miRNA inhibitors, or miR-181b inhibitorsat the final concentration of 50 nM, respectively. After 36 h, cellswere exposed to 10 ng/ml TNF-α for 16 h. Then, the supernatants werecollected for ELISA analysis by means of SearchLight MultiplexImmunoassay Kit (Aushon BioSystems, Inc).

Cell Adhesion Assays

HUVECs grown in 12-well plates were transfected with miRNA mimics orinhibitors. Twenty-four hours later, transfected cells were replatedonto 96-well fluorescence plates (BD, cat#353948) for overnight growth.The following day 10 ng/ml TNF-α was added for 5 h. THP-1 cells (ATCC)were washed with serum-free RPMI-1640 medium and suspended at 5×10⁶cells/mL in medium with 5 μM of Calcein AM (Invitrogen, cat# C3100MP).Cells were kept in an incubator containing 5% CO₂ at 37° C. for 30 min.The labeling reaction was stopped by the addition of the cell growthmedium, and cells were washed with growth medium twice then re-suspendedin growth medium at 5×10⁵ cells/ml. After 5 h TNF-α treatment, HUVECswere washed once with THP-1 cell growth medium. Then 200 μL CalceinAM-loaded THP-1 cells were added to each well. After 1-h incubation,non-adherent cells were removed carefully. Adherent cells were gentlywashed with pre-warmed RPMI-1640 medium four times. Fluorescence wasmeasured by using a fluorescence plate reader at 485 nm excitation. Thenumber of THP-1 cells per view was quantified from randomly acquiredimages.

Real-Time Quantitative PCR

HUVECs were suspended in Trizol reagent (Invitrogen), and total RNA wasisolated according to the manufacturer's instructions. Reversetranscriptions were performed by using miScript Reverse TranscriptionKit from Qiagen (218061). Either QuantiTect SYBR Green RT-PCR Kit(204243) or miScript SYBR Green PCR Kit (218073) from Qiagen was usedfor quantitative real-time qPCR analysis with the Mx3000P Real-time PCRsystem (Stratagene), following the manufacturer's instructions. Gene-and species-specific primers were used to detect human or mouse VCAM-1,E-selectin, PAI-1, and ICAM-1. To amplify mature miRNA sequences,hsa-miR-181a (PN4373117), hsa-miR-181b (PN4373116), hsa-miR-181c(PN4373115), hsa-miR-181d (PN4373180), RNU6B (PN4373381), TaqManMicroRNA Reverse Transcription Kit (PN4366596), TaqMan® Universal PCRMaster Mix, No AmpErase UNG (PN4324018), or miScript primer assays forHs_RN5S1_1 (MS00007574) and Hs_miR-181b_1 (MS00006699) from Qiagen wereused.

Intimal RNA Isolation from Aorta Tissue

Isolation of intimal RNA from aorta was modified from a previous study(Nam et al., Am. J. Physiol. Heart Circ. Physiol. 297:H1535-1543, 2009).Briefly, aorta between the heart and diaphragm was exposed, and theperi-adventitial tissues were removed carefully. The cleaned aorta wascut out and transferred to a 35-mm dish containing ice-cold Hank'sBuffered Salt Solution (HBSS). The tip of an insulin syringe needle wascarefully inserted into one end of the aorta to facilitate a quick flushof 150 μL QIAzol lysis buffer through it and collection of the intimaeluate into a 1.5-ml tube. The aorta leftover (media+adventitia) waswashed once with HBSS and snap-frozen in liquid nitrogen, for storageuntil total RNA extraction by TRIzol.

Immunostaining

HUVECs grown on coverslips were fixed with 4% paraformaldehyde andpermeabilized with 0.5% Triton X-100 (Sigma). After blocking with normalgoat serum, cells were incubated with antibody against p65 followed byCy5-conjugated goat anti-mouse IgG and DAPI (Invitrogen). Images wereacquired with an Olympus Fluoview FV1000 confocal microscope equippedwith a Multi-Ar laser, HeNe G laser, HeNe R laser, and a LD405440 laserdiode. The UPLSAPO 20×NA: 0.75 objective lens was used. The followingparameters were set: Zoom×2, 1024×1024[pixel] image size, C.A. 150 μm.The intensity of p65 nuclear staining was quantified by using BitplaneImaris 6.4.2 software.

MiRNP Immunoprecipitation

MiRNP-IP was performed as previously described (Fasanaro et al., J.Biol. Chem. 284:35134-35143, 2009; Huang et al., Mol. Cell 35:856-867,2009). Myc-tagged Ago-2 (from Cold Spring Harbor, N.Y.) wasco-transfected with either miR-181b or miRNA negative control in HUVECs.Cells were washed in ice cold PBS, released by scraping, and lysed inbuffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 2 mM EDTA, 0.5% Triton X-100,100 units/mL of RNasin Plus (Promega) supplemented with 1× proteaseinhibitor (Roche)). The lysed cell solution was adjusted to a final NaClconcentration of 150 mM prior to centrifugation. One-twentieth of thesupernatant volume was collected in TRIzol for use as an extractcontrol. The remaining portion of the supernatant was pre-cleared withProtein AG UltraLink Resin (Pierce), to which 2 μg anti-c-myc antibodywas added and the mixture allowed to incubate overnight at 4° C.; thefollowing day Protein AG UltraLink Resin was added. After 4 h ofmechanical rotation at 4° C., the agarose beads were pelleted and washedfour times in wash buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.05%Triton X-100). Finally, 1 mL of TRIzol was added into the beads and RNAwas isolated. Total RNA was reverse transcribed into cDNA for real-timeqPCR analysis.

Protein Extraction and Western Blot Analysis

Transfected HUVECs were treated with 10 ng/ml TNF-α for 1 h, thencytoplasmic and nuclear extracts were isolated by NE-PER nuclear andcytoplasmic extraction reagents (Thermo Fisher Scientific). Culturedcells were harvested and lysed in RIPA buffer (50 mM Tris-HCL pH 7.4,150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) supplementedwith complete protease inhibitor cocktail tablets (Roche). To extractprotein, snap-frozen tissues were homogenized in RIPA buffersupplemented with protease inhibitor. Cell or tissue debris was removedby centrifugation at 12,000 rpm for 10 min. Lysates were separated by 8%or 10% SDS-PAGE gels, transferred to PVDF membranes (Bio-Rad), andincubated with the relevant antibodies as where indicated. Proteins werevisualized by ECL Plus Western blotting detection reagents (RPN2132; GEHealthcare). Densitometry scanning of the blots with ImageJ software wasused to calculate the abundance of protein.

Microarray Gene Chip analysis and Bioinformatics

HUVECs were transfected with 10 nM miRNA negative control or miR-181bmimics for 36 h, and treated with 10 ng/ml TNF-α for 4 h. Cells werecollected into TRIzol and sent for Two-Color, 4×44 K format, Human WholeGenome Microarray Service (Miltenyi Biotec Inc.). Differentiallyexpressed genes were identified by using the fold-change lower thresholdof 1.5. Gene set enrichment analysis (Subramanian et al., Proc. Natl.Acad. Sci. 102:15545-1550, 2005) was used to test whether a knownbiological pathway/process or molecular function was suppressed byover-expression of miR-181b. Gene sets used for enrichment analyses weredownloaded directly from the Broad Institute website or the GeneOntology website. Gene sets discovered with a false discovery rate (FDR)less than 25% were considered as significantly enriched. The primersused for real-time qPCR validation are listed in Table 2 below.

TABLE 2 Primers used for qPCR Validation Name Sequence (5′−>3′) BCL2A1-FTACAGGCTGGCTCAGGACTAT (SEQ ID NO: 60) BCL2A1-RTTTTGTAGCACTCTGGACGTTT (SEQ ID NO: 61) C1QTNF1-FCAAGGGAAATATGGCAAAACAGG (SEQ ID NO: 62) C1QTNF1-RATCACCGTCTGGTAGTAGTGG (SEQ ID NO: 63) CCL1-FTCATTTGCGGAGCAAGAGATT (SEQ ID NO: 64) CCL1-RCTGAACCCATCCAACTGTGTC (SEQ ID NO: 65) CCL7-FCCAATGCATCCACATGCTGC (SEQ ID NO: 66) CCL7-RGCTTCCCAGGGACACCGAC (SEQ ID NO: 67) CCR2-FGACCAGGAAAGAATGTGAAAGTGA (SEQ ID NO: 68) CCR2-RGCTCTGCCAATTGACTTTCCTT (SEQ ID NO: 69) CFB-FGCGGCCCCTTGATAGTTCAC (SEQ ID NO: 70) CFB-RCAGGGCAGCACTTGAAAGAG (SEQ ID NO: 71) CSF2-FGGGAGCATGTGAATGCCATC (SEQ ID NO: 72) CSF2-RGCAGTGTCTCTACTCAGGTTCAG (SEQ ID NO: 73) CX3CL1-FACCACGGTGTGACGAAATG (SEQ ID NO: 74) CX3CL1-RCTCCAAGATGATTGCGCGTTT (SEQ ID NO: 75) CXCL1-FAGGGAATTCACCCCAAGAAC (SEQ ID NO: 76) CXCL1-RACTATGGGGGATGCAGGATT (SEQ ID NO: 77) CXCL16-FCAGCGTCACTGGAAGTTGTTA (SEQ ID NO: 78) CXCL16-RCACCGATGGTAAGCTCTCAGG (SEQ ID NO: 79) CXCL3-FCCAAACCGAAGTCATAGCCAC (SEQ ID NO: 80) CXCL3-RTGCTCCCCTTGTTCAGTATCT (SEQ ID NO: 81) CXCL6-FAGAGCTGCGTTGCACTTGTT (SEQ ID NO: 82) CXCL6-RGCAGTTTACCAATCGTTTTGGGG (SEQ ID NO: 83) EGR1-FACCTGACCGCAGAGTCTTTTC (SEQ ID NO: 84) EGR1-RGCCAGTATAGGTGATGGGGG (SEQ ID NO: 85) EGR2-FATCCCAGTAACTCTCAGTGGTT (SEQ ID NO: 86) EGR2-RCTCCACCGGGTAGATGTTGT (SEQ ID NO: 87) EGR3-FGCGACCTCTACTCAGAGCC (SEQ ID NO: 88) EGR3-RATGGGGAAGAGATTGCTGTCC (SEQ ID NO: 89) EGR4-FAGCGAGTTTTCCGAACCCG (SEQ ID NO: 90) EGR4-RGAGTCGGCTAAGTCCCCACT (SEQ ID NO: 91) IL1R1-FACATTGTGCTTTGGTACAGGG (SEQ ID NO: 92) IL1R1-RCCCCAACAGTCTTTGGATACAG (SEQ ID NO: 93) LIF-FGTACCGCATAGTCGTGTACCT (SEQ ID NO: 94) LIF-RCACAGCACGTTGCTAAGGAG (SEQ ID NO: 95) MMP11-FGAGGCCCTAAAGGTATGGAGC (SEQ ID NO: 96) MMP11-RCCCTTCTCGGTGAGTCTTGG (SEQ ID NO: 97) PLAU-FGTGAGCGACTCCAAAGGCA (SEQ ID NO: 98) PLAU-RGCAGTTGCACCAGTGAATGTT (SEQ ID NO: 99) PTGS2-FGTGCAACACTTGAGTGGCTAT (SEQ ID NO: 100) PTGS2-RAGCAATTTGCCTGGTGAATGAT (SEQ ID NO: 101) S1PR1-FCTTGCTGACCATTTGGAAAACC (SEQ ID NO: 102) S1PR1-RCTGTGTAGGCTACTCCTGCC (SEQ ID NO: 103) SDC4-FGCTCTTCGTAGGCGGAGTC (SEQ ID NO: 104) SDC4-FCCTCATCGTCTGGTAGGGCT (SEQ ID NO: 105) SELE-FAATCCAGCCAATGGGTTCG (SEQ ID NO: 106) SELE-R GCTCCCATTAGTTCAAATCCTTCT (SEQ ID NO: 107) SERPINE1-F CATCCCCCATCCTACGTGG (SEQ ID NO: 108)SERPINE1-R CCCCATAGGGTGAGAAAACCA (SEQ ID NO: 109) TIMP3-FCATGTGCAGTACATCCACACG (SEQ ID NO: 110) TIMP3-RACATCTTGCCATCATAGACGC (SEQ ID NO: 111) TNFRSF11B-FAAGGGCGCTACCTTGAGATAG (SEQ ID NO: 112) TNFRSF11B-RGCAAACTGTATTTCGCTCTGGG (SEQ ID NO: 113) TRAF1-FCCGGCCCCTGATGAGAATG (SEQ ID NO: 114) TRAF1-RTTCCTGGGCTTATAGACTGGAG (SEQ ID NO: 115) VCAM1-FGCTGCTCAGATTGGAGACTCA (SEQ ID NO: 116) VCAM1-RCGCTCAGAGGGCTGTCTATC (SEQ ID NO: 117) GAPDH-FATGGGGAAGGTGAAGGTCG (SEQ ID NO: 118) GAPDH-RGGGGTCATTGATGGCAACAATA (SEQ ID NO: 119)In Vivo MiR-181b Over-Expression and Animal Experiments

Male C57BL6 mice 8-10 weeks old were purchased from Charles River. Anequal volume of Atelocollagen (Koken, Tokyo, Japan) and miRNAs (Ambion)were mixed to form complexes, according to the manufacturer's guideline.Each mouse (n=3-6 per group) was administered 200 μL mixtures containing50 μg negative control or miR-181b mimics by tail vein injection.Recombinant mouse TNF-α (2 μg/mouse) from R & D Systems (410-MT/CF) wasintraperitoneally (i.p.) injected on the following day. Four hourslater, mice were sacrificed to harvest tissues and organs for analysis.Lungs were used for Western blot analysis, total RNA extraction, andimmunohistochemistry. Aorta arches were snap-frozen for total RNAextraction, while the descending aorta was fixed with 4%paraformaldehyde and embedded in paraffin for immunohistochemistry.Hearts, spleens, and livers were used for total RNA isolation. Six-μmsections were prepared from paraffin-embedded lung and aorta; sectionswere then deparaffinized with xylene and rehydrated in water through agraded ethanol series, followed by antigen retrieval performed for 5 minin a pressure cooker using Tris-EDTA solution, pH 8.0. The sections weretreated with TBST buffer and 3% H₂O₂, blocked with blocking reagent,incubated sequentially with goat anti-mouse VCAM-1, secondary antibody,and DAB chromagen, and counterstained with hematoxylin. Images wereacquired by a digital system.

For murine endotoxemia model, mice were administered with vehicle, miRNAnegative control, or miR-181b mimics by tail vein injection. On thefollowing day, mice were i.p. injected with 40 mg/kg LPS (Escherichiacoli serotype O26:B6 endotoxin; Sigma-Aldrich) or vehicle (saline). Lungtissues were processed for immunohistochemistry 4 h after LPS treatmentand stained for Gr-1 (BD Pharmingen), CD45 (BD Pharmingen), or VCAM-1.Myeloperoxidase (MPO) activity was measured in lung homogenates usingthe Amplex Red Peroxidase Assay Kit (Invitrogen) according to themanufacturer's instructions. Animals were housed in pathogen-freebarrier facilities and regularly monitored by the veterinary staff.

Statistics

Differences between two groups were examined using the Student's t-test(two-tailed) and were considered significant at P<0.05.

Example 2 Animal Model of Asthma

Asthma is a heterogeneous, complex disorder that involves airflowobstruction, airway hyperresponsiveness (AHR), and inflammation.Accumulating studies highlight a critical role for enhanced NF-κBpathway activation in asthmatic tissues. NF-κB activation leads toincreased expression of adhesion molecules and chemokines on severalcell types, including the lung endothelium. Thus, suppressing theinflammatory response in the pulmonary endothelium may provide a therapyin asthma patients.

An experimental model of asthma in mice will be used to study the effectof nucleic acids containing all or a part of the sequence of miR-181b(SEQ ID NO: 1) on asthma. The nucleic acids described herein, containingall or a part of the sequence of SEQ ID NO: 1, may suppress experimentalasthma. In these studies, ovalbumin (OVA)-sensitized and challenged micewill be injected with one of the nucleic acids described herein (pre- orpost-OVA challenge), and the miR-181b nucleic acid-mediated effects onendothelial cell activation and airway inflammation, airwayhyperresponsiveness to methacholine challenge, expression ofNF-κB-regulated genes, accumulation of eosinophils and leukocyte subsetsin bronchoalveolar lavage fluid (BALF) and lung tissue, CD4⁺ Th2lymphocyte responses, and hypersecretion of mucus will be determined. Anucleic acid containing all or a part of the sequence of SEQ ID NO: 1will be administered either by intravenous injection (tail vein) or byaerosol (nebulization). The nucleic acid containing all or a part of thesequence of SEQ ID NO: 1 may be admixed with cationic molecules such ascollagen (e.g., atelocollagen, or type I telocollagen), lipofectin-basedreagents, lipidoids, elastin-like peptides, small peptides (e.g., a RGDpeptide, PEGylated with RGD, or a collagen), or polymericmicroparticles, or non-cationic molecules, such as PGLA, or aptamersspecific to endothelial cells to enhance systemic intravenous or aerosoldelivery. Alternatively, the nucleic acid containing all or a part ofthe sequence of SEQ ID NO: 1 may be conjugated to molecules such asglucan particles for oral delivery. Effects on the action of NF-κBsignaling in vivo will also be examined using the OVA-challenged,NF-κB-luciferase-GFP transgenic mice and bioluminescence imaging.

Example 3 Animal Models of Atherosclerosis

Arteriosclerosis and its complications are the leading cause ofmorbidity and mortality in Western societies. The lesions ofatherosclerosis represent a series of highly specific cellular andmolecular responses that can be viewed, in aggregate, as an inflammatorydisease process. Experimental, pathologic, and clinic observationssupport a critical role for the activated endothelium in atherogenesis.Activated endothelial cells have been identified in every phase ofatherosclerotic lesion development—from earliest lesion termed the“fatty streak” to the mature and obstructive plaque. NF-κB activationleads to induction of adhesion molecule expression in endothelial cells,an effect that leads to leukocyte accumulation within the vascular walland atherosclerotic lesion formation.

An experimental model will be used to examine the effect of a nucleicacid containing all or a part of the sequence of SEQ ID NO: 1 onatherosclerosis initiation and progression in vivo. In the proposedstudies, mice will be intravenously injected with a nucleic acidcontaining all or a part of the sequence of SEQ ID NO: 1 and controls,at least weekly in: atherosclerotic-prone LDL-R-deficient orApoE-deficient 8-10 week old mice fed a high fat diet for 8 weeks(initiation studies), or after atherosclerotic-prone LDL-R-deficient orApoE-deficient mice have already been fed a high fat diet for at least 8weeks (progression studies). The severity of atherosclerotic lesionformation and the cellular composition of the lesions in the aorticsinus and descending aorta will be determined using standardimmunohistochemical techniques.

Example 4 Animal Models of Obesity

Obesity contributes to a significant health epidemic and is a major riskfactor for coronary artery disease and diabetes, and other chronicinflammatory disease states, such as degenerative neurological disease,lung airway disease, diabetic retinopathy, and cancer. Indeed, almosttwo-thirds of American adults are overweight. Obesity is associated withchronic inflammatory signaling pathways, such as NF-κB. Clinical andexperimental studies indicate that activation of the NF-κB signalingpathway can increase insulin resistance and type 2 diabetes. Thus,targeting the NF-κB signaling pathway may offer a strategy for treatingobesity and decreasing cellular inflammation.

The effect of a nucleic acid containing all or a part of the sequence ofSEQ ID NO: 1 on obesity and insulin resistance will be studied usinganimal models. In these experiments, a nucleic acid containing all or apart of the sequence of SEQ ID NO: 1 will be injected intravenously atleast weekly in one or more of four models of obesity for a total offour weeks: Zucker fatty rats, ob/ob mice, db/db mice, and C57BL6 micefed a high-fat diet. Blood glucose and insulin concentrations will bemeasured during glucose tolerance testing. Insulin sensitivity will bemeasured using insulin tolerance tests. Lipid profiles (cholesterol,LDL, triglyceride, and HDL) and free fatty acid levels will also bemeasured. Finally, body composition will be analyzed including visceraland subcutaneous fat profiles. The activity of NF-κB (e.g., expressionof NF-κB target genes) will be examined in fat, liver, and skeletalmuscles. Finally, body weights, energy expenditure, and caloricconsumption will be analyzed. A nucleic acid containing all or a part ofthe sequence of SEQ ID NO: 1 may improve insulin sensitivity, reduceglucose levels, reduce free fatty acid levels, and improve body weightand composition.

Example 5 Animal Models of Rheumatoid Arthritis (RA)

Rheumatoid arthritis is a systemic chronic inflammatory disease thatprimarily attacks the synovial joints, an effect leading to destructionof articular cartilage and joints with considerable disabling, painfulmorbidity. Indeed, about 1% of the world's population is affected by RA.Clinical and experimental studies indicate that activation of NF-κBsignaling pathway is one of the principal targets in RA. NF-κB promotesthe accumulation of leukocytes, abnormal growth of fibroblast-likesynovial cells, and is involved in the differentiation and activation ofbone-resorbing activity of osteoclasts. Current drug therapies, such asTNF-α antagonists, such as etanercept, infliximab, or adalimumab, orIL-1 antagonists, such as anakinra, block activation of NF-κB signalingand RA disease progression. However, side effects may be associated withthese drugs. Furthermore, downstream targeting of TNF-α or IL-1 mayprovide a more potent and specific method of reducing NF-κB activation.Thus, targeting the NF-κB signaling pathway may offer novel strategiesfor limiting RA disease progression.

The effect of a nucleic acid containing all or a part of the sequence ofSEQ ID NO: 1 on rheumatoid arthritis is being studied in an animalmodel. In these experiments, a nucleic acid containing all or a part ofthe sequence of SEQ ID NO: 1 is injected intravenously at least weeklyin three models of RA: rat adjuvant arthritis, rat type II collagenarthritis, or mouse type II collagen arthritis. For the adjuvant model,treatment is administered either at day 0 (prophylactic model) or day 8(therapeutic model). The study is terminated on day 15. The tibiotarsaljoint and foot paws are examined for inflammation and arthriticparameters. For the rat type II collagen arthritis model, rats are giventype II collagen (2 mg/mL in incomplete Freund's adjuvant) on day 0 andday 7. Onset of arthritis occurs on days 10-13. Rats are harvested forhistopathological evaluation at the completion of treatment. For themouse type II collagen model, DBA1 lacJ mice are immunized againstbovine type II collagen at day 0 and day 21 with and without concurrentboosting with endotoxin or recombinant IL-1. The disease occurs inpaws/joints and measurements of disease are determined clinically andwith histological scoring. NF-κB activity (e.g., the expression of NF-κBtarget genes) is examined in tissues. This model has been used toevaluate effects of IL-1 and soluble TNF-α receptor antagonists. Bothprophylactic and treatment studies are performed using a nucleic acidcontaining all or a part of the sequence of SEQ ID NO: 1. A nucleic acidcontaining all or a part of the sequence of SEQ ID NO: 1 may inhibitNF-κB activity in tissues and may reduce RA disease.

Example 6 Animal Models of Crohn's Disease and Ulcerative Colitis

Crohn's disease and ulcerative colitis are common inflammatory boweldiseases that can affect portions of the gastrointestinal tract and cancause significant symptoms and morbidity. It is estimated that ˜1million people in the U.S. are affected by inflammatory bowel disease(IBD). Treatment options are quite limited to maintaining remission,preventing relapse, and controlling symptoms. Clinical and experimentalstudies indicate higher levels of expression of NF-κB signaling pathwayin biopsy samples from patients with IBD. Immunomodulating agents, suchas corticosteroids, which reduce NF-κB activation, are helpful inpatients, but have numerous side effects. Thus, targeting NF-κBsignaling pathway may offer novel strategies for limiting IBD diseaseprogression.

The effect of a nucleic acid containing all or a part of the sequence ofSEQ ID NO: 1 on IBD is studied using an animal model. In theseexperiments, a nucleic acid containing all or a part of the sequence ofSEQ ID NO: 1 is injected intravenously at least weekly in three modelsof IBD in mice: chemically-induced IBD, genetically-modifiedIL-10-deficient or TNF delta ARE mice, or spontaneous IBD strains ofmice (SAMP1/Yit). For the chemically-induced model, dextran sodiumsulfate (DSS) is used, which is well-characterized for inducingepithelial disruption resulting in bacterial and neutrophilinfiltration, and represents a model of the acute phase of injury.IL-10-deficient mice have a transmural colitis and are useful to assessthe role of specific cytokine signaling pathways. TNF delta ARE micerepresent a model of Crohn's-like disease with ileal involvement. Thisis an important model as TNF-α contributes to the pathogenesis ofCrohn's disease and up to 70% of patients with refractory Crohn'sdisease respond to TNF-α blocking treatment. The SAMP1/Yit model ofileitis also provides a stable model of IBD and has implicated vascularadhesion molecules, such as VCAM-1, as being important for leukocytetrafficking and accumulation in IBD development. Indeed, blockingantibodies to VCAM-1 and ICAM-1 leads to a 70% improvement in severityof inflammation. For each of these models, a nucleic acid containing allor a part of the sequence of SEQ ID NO: 1 is intravenously injected atleast weekly in: prophylactic studies (early in the course of disease,e.g., 4-6 weeks), or treatment studies (after established IBD isobserved (varies between 8-20 weeks depending on the model)). Theseverity of IBD and the cellular composition of the gastrointestinaltract is determined using standard immunohistochemical techniques. Anucleic acid containing all or a part of the sequence of SEQ ID NO: 1may inhibit the progression of IBD.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

What is claimed is:
 1. A method of treating lung inflammation or asthmain a subject, the method comprising: identifying a subject in need oftreatment for lung inflammation or asthma; and administering to thesubject a therapeutically effective amount of a nucleic acid comprisingSEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3, wherein the nucleic acid has120 or fewer nucleotides in length.
 2. The method of claim 1, whereinsaid nucleic acid is conjugated to one or more of a polymer, a peptide,and a polysaccharide.
 3. The method of claim 1, wherein said nucleicacid is conjugated to a lipid moiety.
 4. The method of claim 1, whereinthe nucleic acid is delivered in liposomes.
 5. The method of claim 1,wherein the nucleic acid is administered intravenously.
 6. The method ofclaim 1, wherein the nucleic acid is delivered by aerosol.
 7. The methodof claim 1, for treating lung inflammation in a subject, the methodcomprising identifying a subject in need of treatment for lunginflammation.
 8. The method of claim 7, wherein the lung inflammation isassociated with acute lung injury.
 9. The method of claim 1, fortreating asthma in a subject, the method comprising identifying asubject in need of treatment for asthma.